Category: blogpost

The paper strip test was developed at the Council of Scientific and Industrial Research’s constituent lab, the Institute of Genomics and Integrative Biology (CSIR-IGIB) based in New Delhi, India. The testing kit addresses the urgent need for accurate COVID-19 mass testing and has many advantages compared to the gold standard, qPCR. Like SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) which is a COVID 19 test using Cas12 for detection of SARS-CoV-2, developed by scientists from the Broad Institute in the US, the name Feluda refers to an Indian fictive detective (film director: Satyajit Ray).

Advantages of the Feluda test compared to qPCR:

# Affordability
# Can be used in settings with limited resources
# Less time to result (around 45 minutes)
# No qPCR equipment (no need for expensive devices)
# High ease of use
# Lateral Flow based readout for easy interpretation of results

With 96% sensitivity and 98% specificity, Feluda’s analytical performance is comparable to the qPCR results.

How the Feluda test works

The COVID-19 test developed in New Delhi, India by a research team led by Debojyoti Chakraborty and Souvik Maiti is based on a Cas9 enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity. Just a few weeks ago, Emmanuelle Charpentier and Jennifer Doudna have been awarded the 2020 Nobel Prize in Chemistry for their development of CRISPR/Cas9 gene editing technology. The Cas9 readout used for coronavirus detection has requirement of trans-cleavage activity of reporter molecules like Cas12 or Cas13 methods.

The scientists from CSIR-IGIB used Cas9 from Francisella novicida (FNCas9), which shows very high mismatch sensitivity and can distinguish between nucleotide sequences differing by only one mismatch. The principle of the paper strip test was originally designed for the identification of sickle cell anemia, a disease caused by a point mutation, and adapted to COVID-19 testing due to the Coronavirus outbreak resulting in an urgent need for mass testing.

The FnCas9 used in this new method does not produce collateral activity on substrates due to the use of a catalytically inactive FnCas9-gRNA-complex. Therefore, it is an affinity based method and no trans-cleavage signal output is generated.

Fig. 1: Feluda test principle on HybriDetect. FnCas9 with FAM labeled gRNA binds to biotinylated sample if result is positive.

The Feluda test and Milenia HybriDetect

The indian researchers designed a gRNA, labeled with FAM, which is important for the visualization of the test line on Milenia HybriDetect (Figure 2).

After RNA extraction, the first step of the paper strip test is an optimized single step Reverse Transctription-PCR (RT-PCR), or alternatively a RPA protocol, where the sample gets amplificated and biotinylated.

In the next step, the FnCas9 gRNA (labeled with FAM) is incubated with the biotinylated substate (virus sequence if sample is positive). Due to the streptavidin, immobilized on the HybriDetect dipstick, the RNP Complex (CRISPR ribonucleoprotein) binds to the test line (Figure 2).

The RNP-complex bound to the labeled substrate is visualized with anti-Fam antibodies conjugated to gold nanoparticles and a positive test line is formed if substrate is bound to the RNP-complex. Summarized this means, two lines for a positive and a single line for a negative test result. The visual interpretation of the HybriDetect dipstick is as easy as the interpretation of a pregnancy test and therefore no trained personnel is required.

Fig.2: Milenia HybriDetect and FnCas9. The test is positive if the biotinylated DNA is present and binds to streptavidin on the T-Line. FnCas9 binds to the biotinylated DNA and is captured by FAM antibodies coated with gold nanoparticles for visualization.

When to expect Feluda Test to be available?

The Union Health Minister of India Harsh Vardhan said on October 11th, that the rollout of the new Covid-19 test is expected in the next few weeks. The test developed by scientists from the Council of Scientific and Industrial Research’s Institute of Genomics and Integrative Biology in New Delhi is going to provide results in 45 minutes and is priced around Rs 500 ( ~ 5,70 Euro).
Additional Information on the Feluda test

# Compared to other CRISPR methods for COVID Detection, including SHERLOCK and DETECTR, Feluda does not need a reporter and is therefore less complex.
#  The LOD of indias new test method is 10 copies of purified viral sequence as the authors mention in their latest publication (https://www.medrxiv.org/content/10.1101/2020.09.13.20193581v1.full.pdf)
#  To assist the detection of COVID-19 the scientists developed a smartphone app named TOPSE (True Outcome Predicted via Strip Evaluation) which gives a predictive score based on background correction. Due to the stoichiometric binding affinity of FnCas9 RNP to the target, a semi-quantitative readout of Feluda is possible with TOPSE.

Feluda for at home testing

The Scientists from CSIR-IGIB are currently working on a Feluda version for at home coronavirus testing. Therefore they are trying to do a machine free, RPA based amplification of the virus genome. The aim is an end-to end instrumentation free testing protocol.

Contamination in amplification steps can occur from many reasons

The general reason for contamination is the presence of templates and amplicons in the lab environment. Most labs are working with several employees on one topic for some months or years, for example an inherited disease, where a specific gene plays a major role. You can imagine, contamination of the lab environment with this specific gene can easily occur (I had this problem with GFP, which everyone used as reporter gene during my phD).

Finding the source of a contamination is like looking for a needle in a haystack. To name a few possibilities: check/clean/exchange your pipettes, the centrifuge, the bench top, vortex, racks, tips, reagents (polymerase, buffer, nucleotides, water,…).

General Lab Practice to avoid Contamination

Most people (as so do I) working with DNA amplification methods know the terrible moment, seeing a positive signal in your negative control. First, you still have a faint hope: “Maybe I was not concentrated and put some template DNA instead of Water into my negative control”

After repeating the experiment and seeing again false positive results, one has to do a lot of troubleshooting.

To help you, prevent this time wasting, annoying work I want to give some general advices:

1. My first recommendation is to spatially separate the two main steps of your experiment: a) Preparation of the PCR/LAMP/RPA reaction and b) the amplification step and lateral flow readout. The mastermix should additionally be prepared in a template-free area of your lab. If you have enough space, go to another room to do the different steps. When changing the room, don’t forget to change your gloves and lab coat. Even if it is time consuming and annoying, think of the time you might lose, if you have a cross-contamination! Don’t go back to the preparation area with your samples if you already have been in the amplification area, you always have to work in one direction.
2. I know, most people working in the lab have special preferences when it comes to pipettes, sometimes it’s like a superstition. But I will recommend you not to use the same pipette for the different steps of your Experiments. You need at least one or more (depending on the volumes) pipette(s):
   * for the preparation of your mastermix
   * for the addition of the template DNA
   * for the transfer to the LFA

To further avoid contamination through pipetting: use filter tips.

1. Same thing for reagents: use dedicated reagents (e.g. water) for the different steps/areas
2. Try to prevent aerosols as much as you can: Spin all tubes before opening!
3. Don’t forget to change your gloves between the different steps.
4. If you have the chance, use a uv sterilizer for the area where your mastermix is prepared
5. Use a clean (autoclaved) rack
6. Use nuclease free water, which is purified, double-distilled, deionized and outoclaved.

What to do, if you already have contamination in your amplification experiments?

First of all, change all reagents. If possible, go to another lab with your fresh reagents and check if there is still contamination. Don’t bring anything except the reagents! Do not bring your pipettes!

If there is still contamination, check your reaction. It might be that your primers are non-specifically binding: Design a new set of primers.

Check, if you are doing the 8 steps I mentioned above. You can try to change your pipettes, use filter tips and so on.

Another possibility is to use UNG (uracil-N-glycosylase). You have to use dUTP instead of dTTP in your amplification reaction. Carryover contamination from amplification products can then be prevented by adding UNG to your reaction and incubate it (prior to amplification) for 2 minutes at 50°C. UNG degrades products that have already been through the amplification process by removing Uracil. The polymerase and the other reagents are not affected by the UNG treatment, only carryover products will be removed.

Fig. 1: Contamination in Lateral Flow Assay. Negative sample shows false positive result due to contamination. Comparison of results withour UDG (upper picture) and with UDG (lower picture)

Question: What is the limit of detection of Milenia HybriDetect in pmol?

Over the many years of being in the market with our development tool, we got quite a lot of questions from our customers. Some of these questions are frequently raised. The answers to these questions are given on the HybriDetect Landingpage.

Other questions may be raised individually, but we try to respond to every individual question with the best of our knowledge. If we do not know the answer right now, we run experiments in order to supply our customers with an adequate answer. Just recently a customer wanted to know what the limit of detection of Milenia HybriDetect in pmol is!

Customer reports on the sensitivity of Milenia HybriDetect

To answer this question we did a little literature search and assorted several papers dealing with information about the limit of detection of our HybriDetect.

Piepenburg et al. (2006)

In the very first publication on RPA amplification Piepenburg et al. (2006) used Milenia HybriDetect to detect amplicons. They introduced samples with 10 copies each of MRSAIII, MRSAII and MRSAI and 10.000 copies of MSSA (negative control) as a template in their MRSA assay. The test line was clearly visible on all HybriDetect lateral flow strips with MRSA samples, indicating that the sensitivity of the system must be between 1 and 10 copies of the target gene.

Kiatpathomchai et al.. 2018

In 2008 a paper on “Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick” was published in the Journal of Virological Methods by

Kiatpathomchai et al..

They found out that the combination of their LAMP method and Milenia HybriDetect is overall 100 times more sensitive than an agarose gel and 10 times more sensitive than a nested PCR followed by electrophoresis. Positive side effects of the use of a lateral flow strip were the elimination of a potentially hazardous compound like ethidium bromide, the spimplicity in use and the choice of a true point-of-need application.

Pecchia and Da Lio (2018)

In 2018 Pecchia and Da Lio published a paper with the title “Development of a rapid PCR-Nucleic Acid Lateral Flow Immunoassay (PCR-NALFIA) based on rDNA IGS sequence analysis for the detection of Macrophomina phaseolina in soil”.

The authors found out that the limit of detection of the NALFIA assay was 17.3 fg using 10 μl PCR reaction. In contrast to this the sensitivity of the agarose gel electrophoresis was 17.3 pg using 25 μl of PCR reaction. This means that in the system of Pecchia and Da Lio the sensitivity of Milenia HybriDetect was 1.000 fold higher than the agarose gel.

However, it needs to be stressed that the advantage of Milenia HybriDetect compared to an agarose gel in terms of sensitivity depends on the size of the amplicons to be detected. Pecchia and Da Lio created amplicons of 100 bp length!
The Limit of Detection of HybriDetect can be as low as 0,1 fmol

Beside the literature search, we did experiments to find out the limit of detection of HybriDetect in our lab. Therefore a dual labeled dsDNA (Biotin/FITC), generated by a PCR was transferred to Milenia HybriDetect strips and run. The concentration of the samples ranged from 0 to 250 fmol. The results of the experiment are shown in Figure 1.

Figure 1.Limit of Detection (lower limit) of HybriDetect (MGHD1) and HybriDetect2T (MGHD2).[T1] test line of the MGHD1 and MGHD2. [T2] test line 2 is a special feature of the MGHD2. Analysis was performed with a dual labeled PCR-product (Biotin and FITC: T1, Digoxygenin and FITC: T2).

By using dual labeled dsDNA the LOD was calculated below 1 fmol. This approach is limited in terms of precision due to the purification of the PCR fragments, the measurement procedure and the high dilution of the samples.

Therefore, a different approach was done and a synthetic reporter gene (dual labeld ssDNA, very short oligos) was tested with HybriDetect. The results indicate that 0,5 fmol of the reporter resulted in clear signals on the T-line of the Milenia HybriDetect test strip. Very slight signals were visible by using the reporter below 0,05 fmol. (Figure 2).

Figure 2. Lower detection limit of HybriDetect (MGHD1). [T] test line of the MGHD1. Analysis was performed with a dual labeled Reporter (Biotin and FITC: T).

Conclusion

Milenia HybriDetect is a lateral flow test development platform based on a universal test strip. The product is frequently used in molecular biology applications and customers are interested to know how sensitive their assays can be! As a result of a literature search and own experiments it was demonstrated that Milenia HybriDetect could be up to 1.000 times more sensitive than the detection of amplicons via an agarose gel. The sensitivity of HybriDetect could go down as much as 0,05 fmol.

Literature:

1. Piepenburg O., Williams C.H., Stemple D.L., Armes N.A. DNA detection using recombination proteins. PLoS Biol 2006; 4(7): e204. DOI:10.1371/journal.pbio.0040204
2. Kiatpathomchai W., Jaroenram W., Arunrut N., Jitrapakdee S., Flegel TW. Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. Journal of Virological Methods 2008; 153: 214–217
3. Pecchia S. and Da Lio D., Development of a rapid PCR-Nucleic Acid Lateral Flow Immunoassay (PCR-NALFIA) based on rDNA IGS sequence analysis for the detection of Macrophomina phaseolina in soil. Journal of Microbiological Mehtods (2018, Vol 151)

There are two variants of light chains: kappa and lambda. Regarding the light chains one variable and one constant domain exist.In contrast the heavy chains have one variable and three to four constant parts. These constant parts determine the isotype classification. As most other vertebrates, mice have five antibody-isotypes (IgG, IgM, IgA, IgD, IgE) The main antibody subclasses are formed at different stages of the immune response and have different responsibilities. The IgG isotype is once more divided into IgG1, IgG2a, IgG2b, IgG2c and IgG3 (subtypes) because of sequence varieties between the constant regions (depending on mouse strain).

Mouse Antibody Isotyping Kit – Application

The Mouse-Isotyping Kit is a lateral flow immunoassay designed for the determination of mouse monoclonal antibody isotypes, subtypes and light chains within 5 minutes. The immunoassay can be used for cell culture supernatants and/or purified antibodies.

The Mouse Isotyping Kit consists of a Universal Module (the lateral flow strips) and nine specific buffers, which you can choose according to your needs.

Beside the pure identification of antibody isotypes and subtypes, the Milenia Quick Line Mouse Isotyping Kit has many more options for possible applications and Advantages:

◉ Variableapplication: You can chose which isotypes you need to test – the dipstick is universal, the buffers for the different isotypes, subtypes and light chains can be ordered separately.
◉ Fast IgM selection: IgM antibodies can be quickly detected and selected in an early stage of hybridoma production, as IgMs are in most cases undesired when producing monoclonal antibodies.
◉ Fast, easy and low priced application to find out which cell clone produces antibodies, only one drop of cell culture supernatant is needed.
◉ Recloning advantage: if there is only few/expensive antigen available, you can search for positive clones as an easy preselection with this test system.
◉ Purification method: the optimal purification method can be chosen for the specific isotype which results in the highest yield of the antibody.
◉ Purification control: When purifying the cell culture supernatant, you can quickly detect the desired antibody in the individual fractions.
◉ Semi-quantitative detection: If you apply a standard curve with a known amount of antibody, you can check the concentration of antibody in your sample within minutes.
◉ Purity check: You can see if your antibody is clean or if you have any impurities (other subclasses, e.g.: when your antibody is not monoclonal).
◉ Field of application: The assay can be used for cell culture supernatants and/or purified antibodies.
◉ Selection of secondary antibody: Knowing the isotype of the primary antibody can help detecting the right secondary antibody in immunoassay applications.
◉ IgG2c and IgG2a detection: we offer the possibility to discriminate between IgG2a and IgG2c isotypes, which can be important for the selection of the optimal secondary antibody. In other test systems, IgG2c antibodies are often false identified as IgG2a.

Test principle of the Mouse Isotyping Kit

First the isotype-specific Sample buffer (e.g. anti-mouse IgG1) is pipetted into a well of a 96-well microtiter plate. Subsequently the test sample of unknown antibody-subclass is added. If a complex is formed between the Sample buffer and the test sample, binding to an anti-mouse antibody on the universal mouse test strip will result in the formation of a red line.

Antibody Isotype detection in three simple steps. First, the specific antibody Buffer is added in a well. Second, the sample to be analysed is added to the Buffer. Third, the universal dipstick is added. The readout can be performed after 5 minutes of incubation.

Fig. 1: HybriDetect 2T principle with PCR sample. labeled PCR amplificate can bind to the test line on the strip using biotin label. Bound sample gets visible through gold nanoparticles, which bind the amplificate via FITC

More sensitivity with higher Volume?

Although PCR amplificates are already good to evaluate with the LF strip, there still is potential for an even more sensitive detection. Currently, the optimal application volume for the LF evaluation is only 2 μl out of the total of 25 μl PCR product. If a larger volume is applied, the intensity of the signal does not increase, as one would presume, but decrease (see fig. 2). This means that only a small fraction of the amplified fragment is actually used for detection. Therefore the evaluation could be much more sensitive, if it would be possible to use the entire amount of PCR product.

Fig.2: Applying larger Volume leads to a loss of sensitivity. Signal intensity of HybriDetect 2T with 2 µl, 10 µl and 25 µl of PCR product.

Free primers causing loss of sensitivity

The loss of sensitivity mentioned above can be explained as follows: To receive as much amplificate of the original DNA sequence as possible, the marked primers are added in excess to the PCR mix. Hence, some free primers, which are not used in the reaction, remain in the PCR product. Due to their markings, these free primers are able to bind to the binding sites of the lateral flow strip (and the binding site of the chromophoric gold conjugate) in competition to the double stranded PCR amplificate as shown in figure 3. Regarding the FITC marked primer, this means that the primer is able to bind onto the anti-FITC antibodies loaded with gold particles in the same way as the PCR product that is to be detected, so that the resulting signal of the PCR product is attenuated.

Fig.3: Loss of sensitivity due to competetive reaction. Comparison of the signal intensities of the ideal case evaluation and the real evaluation, containing the competetive reaction between free FITC labeled primers and PCR product.

Designing block oligos to overcome the loss of sensitivity

An idea to compensate the attenuation of the signal and thus loss of sensitivity caused by the free primers, is reflected in the design of extended and modified antisense sequences of these primers, so-called block oligos. The block oligos consist of the antisense sequence of the corresponding, a few spacer nucleotides, a hairpin structure and additional nucleotides serving as overhang (see fig. 4).

Fig.4: Structure of block oligos to overcome loss of sensitivity. Antisense sequence (blue) bind to free detection primer. Spacer (orange), hairpin (red) and overhang (green) ensure that FITC label (pink) of the detection primer (gray) is shielded.

The idea behind this design is that when these block oligos are added to the PCR mix, after the PCR but prior to the evaluation, they bind to the remaining free primers because of the antisense sequence and the remaining structural elements of the block oligo mask the FITC label of the free primers so that the free primers bound to the block oligos can no longer be bound to the gold conjugate.tag verwenden

Effect of the designed block oligos

To test the effect of the designed block oligos, a test system was developed in which the signal intensity of the block oligo sample was compared with the maximum possible signal intensity (artificial sample without free FITC labeled primers) and with the currently achievable signal intensity. By adding the block oligos to the PCR product and evaluating 25 μl instead of only 2 μl amplificate, a tripling of the signal intensity and thus a tripling of the sensitivity could be reached, as shown in figure 5.

Fig 5.: Effect of the block oligos. Relative signal intensity of the various evaluation variants.

Summary

Regarding the evaluation of PCR amplicons, it was found that the free detection primers, the FITC labeled primers in particular, are responsible for a loss of sensitivity in the evaluation. These free primers can bind to the gold conjugate of the evaluation strips in competition to the PCR amplificate. An improvement for this problem could be achieved with the help of so-called block-oligos, which are added to the PCR product together with the running buffer and whose structure allows them to bind to the free primers and shield their FITC labeling.

The assay is based on the SHERLOCK method, combined with Milenias Lateral Flow Assay HybriDetect, which gained lots of interest in the last weeks due to its ability for Covid-19 detection (read more). SHERLOCK is a CRISPR based method, if you want to get an insight on CRISPR and how it works you can read this article for more information.

Detection of three different parameters

The new diagnostic test for post-transplantation Monitoring is able to detect two common opportunistic viruses, which can lead to graft rejections. In addition, the expression status of a special mRNA that serves as a biomarker for rejection can be examined.

Cytomegalovirus (CMV): CMV-infections are usually not assiciated with symptoms in healthy individuals, but can cause serious illness (even fatal) in patients after organ transplantation.
BK polyomavirus (BKV): After kidney transplantation, the BKV can cause a gradual deterioration in graft function and ultimately even graft loss.
CXCL9 mRNA: The expression of CXCL9 increases during rejection in renal-transplant recipients, therefore CXCL9 mRNA is a useful biomarker to identify refection episodes in kidney transplant patients.

Point of Care -Diagnosis with the help of SHERLOCK

Principally, the CRISPR-based method SHERLOCK is capable to detect almost every viral infection. For the assay, described by Kaminski et al. DNA from uninfected and infected patients was isolated from blood and urine. Conserved regions of CMV and BKV were amplified by Recombinase Polymerase Amplification (RPA). A guide RNA for Cas13 complementary for the RPA product was generated. The nuclease activity of the CRISPR/Cas-complex is activated in presence of the transcribed amplificate. Nuclease-dependent reporter degradation is detectable with the HybriDetect test strips.

31 urine and 36 plasma samples from patients were tested for BKV and CMV. BKV could be detected with 100% sensitivity and specificity. CMV was also detected with high sensitivity and specificity in plasma.

For CXCL9 mRNA detection, RNA was isolated from pelleted urine cells. A preamplification is necessary for a sensitive analysis. Therefore, a one-step RT-PCR was performed. After T7-transcription, Cas13-associated RNA recognition initiated collateral nuclease activity of the CRISPR/Cas-complex. Degradation of a dual labeled reporter leads to increasing test line intensity in the LFA. The Cas13-associated detection method was able to detect CXCL9 mRNA at attomolar level. The detection of kidney rejection was shown with a sensitivity of 93%.

Fig. 1: Post-transplantation monitoring of CMV and BKV with SHERLOCK from human urine and blood samples

Detection of SHERLOCK results by HybriDetect

The authors used Milenias HybriDetect for a fast and easy, but still cost effective readout of their results. With HybriDetect it was possible to see a positive or negative test result within two hours from isolation to detection. Some background noise leaded to a faint test band, and therefore the readout of the assay was difficult at very low levels of target concentration. To work around this background noise, Kaminski et al. developed a smartphone-based software application. This app is able to quantify the band intensities of HybriDetect and gives a valid test result in 1 minute (Fig. 2).

Fig. 2: Detection of SHERLOCK results by Lateral Flow readout

Ultrasensitive detection using SHERLOCK and HybriDetect

CMV and BKV (Fig. 3) were detected at different concentrations in patient samples.BKV infection in a kidney-transplant recipients could be successfully identified with the CRISPR-LFA. After the patient was treated, BKV was not detectable.

The authors also tested the lateral flow signal variability over time. The same ten BKV-positive or negative patient samples were tested on three different days. There was no variety in the results over time.

Kaminski et al. compared different incubation times and temperatures for the lateral flow assay and showed that reaction time and temperature are important variables if quantitative lateral flow readout is done. For more information on this topic see our Blog Article: Lateral Flow Readout for CRISPR/Cas-based detection strategies.

Fig. 3: Detection of CMV and BKV in patient plasma samples with HybriDetect and a smartphone app

Beside the detection of viral DNA, HybriDetect was also able to detect CXCL9 synthetic RNA down to the attomolar range. The detection method was confirmed by testing samples from two patients. In one patient, CXCL9 mRNA was detected during rejection, after treatment the test was negative. The other patient also showed a reduced CXCL9 mRNA level after treatment, but an increased amount 7 month later. Repeated Biopsy showed a chronic active cellular rejection.

Overall this work showed a great advantage when it comes to POC testing for post-transplantation Monitoring. The main advantages are the fast and cost effective tests to enable early diagnosis and monitoring after organ transplantation.

Read the original article here

Reference:

Kaminski et al., 2020. A CRISPR-based assay for the detection of opportunistic infections post-transplantation and for the monitoring of transplant rejection.

More information:

https://www.mdc-berlin.de/news/press/diagnostics-meet-crispr

https://static-content.springer.com/esm/art%3A10.1038%2Fs41551-020-0546-5/MediaObjects/41551_2020_546_MOESM3_ESM.mov

As shown in Figure 1, the interpretation of the test strips can vary using different detection strategies. Compared to the HybriDetect instructions for use, the T- and C-lines are swapped in CRSIPR/Cas-associated detection strategies. This leads to a completely different assessment of the resulting signals. The following article is intended to explain this discrepancy in detail and it provides some important information on how to use the HybriDetect in combination with the SHERLOCK / DETECTR method.

Figure 1. Difference of test result interpretation between the HybriDetect Instructions for use and the SHERLOCK-/DETECTR-protocol guidelines

What defines the control- and test-lines?
The “T and the “C” in Lateral Flow Devices (LFDs)

The presence of at least one visually detectable test-line (T-line) is the essential feature of LFDs. In most cases the presence of the T-line correlates with a positive test result. Additionally, a control-line (C-line) confirms the general functionality of LFA. This is absolutely necessary for differentiation between valid negative results and invalid, non-interpretable test results.

The HybriDetect design is basically comparable to the structure of a classic LFD, but the idea behind this detection platform differs.

Figure 2. Composition of the HybriDetect. Biotin and FITC/FAM are introduced into LFA through sample application. The control-line (C) will show up, if you run the HybriDetect without sample. GNP – Goldnanoparticle

HybriDetect and signal generation strategies

The idea behind the HybriDetect platform differs fundamentally from “classic” LFAs. The HybriDetect and HybriDetect 2T are universal test strips, developed to work in multiple detection concepts and not in just one setting. They can be used to detect amplicons, proteins, ribosomal RNAs, whole cells or metabolites. Over the years various detection strategies have been developed, in which the HybriDetect is used.

In most constellations, biotin and FITC are somehow incorporated into analytes or reaction products, which results in the formation of the predefined test-line(s). Popular applications combined with HybriDetect readout are especially DNA amplification techniques such as PCR, RPA or LAMP. However, these LFA-compatible methods have one feature in common: they are designed according to the described Label Incorporation Strategy.

Figure 3. Overview of two LFA-related signal generation stratefies. Label incorporation strategy leads to Test-line formation, whereas reporter degradation will end up in decreased T-line and increased C-line intensity.

In contrast to this, methods such as SHERLOCK or DETECTR generate amplification products, which do not directly contribute to signal generation. Rather, CRISPR complexes are able to specifically and sensitively recognize defined amplicon-related sequences. Successful recognition leads to the activation of the Cas-protein associated collateral nuclease activity. The ssRNA- / ssDNA- / dsDNA – degrading activity is the basis for signal generation in LFA. This is achieved by using the reporter degradation strategy.
The inverse approach – degradation of the “original” T-line

The key to the functionality of a CRISPR-associated nuclease assay combined with a lateral flow readout is the use of a so-called reporter. Such reporters are short single-stranded DNA- or RNA-Oligos with terminal biotin- and FITC-/ FAM-modifications. Reporter sequences are chosen according to cleavage preferences of the used Cas-protein. The following table shows a brief overview of some reporters, which are used in the most common CRISPR/Cas-based detection systems.

Table 1. Overview of reporter sequences used in CRISPR/Cas-based detection methods combined with Lateral Flow readout

An intense test-line appears if a defined amount of reporter is analyzed with the HybriDetect. But if reporter degradation is initiated due to the onset of nuclease activity, the intensity of the T-line consequently decreases.

If the reporter is used in a very defined concentration, the majority of the gold conjugate is trapped at the T-line, which leads to an almost complete extinction of the C-line. It is precisely this effect, that enables the T- and C-line swap for CRISPR/Cas-associated detection methods. Ultimately, it’s just a matter of definition and there is no change in HybriDetect composition! The following figure explains the CRISPR/Cas-based detection strategy with newly defined T- and C-line.

Figure 4. Switching C- and T-line: General mechanism of CRISPR/Cas-mediated detection of nucleic acids via HybriDetect Lateral Flow. (A) The presence of the genetic target leads to a positive test result. (B) The absence of the specific genetic target leads to a negative test result.

Switching test- and control-line has two major advantages: An increasing or appearing signal correlates with a positive test result. Negative results are interpreted if no signal appears. This positive correlation is much more logical for most people and avoids intuitive misinterpretation. This is a very important point when it comes to a point-of-care-compatible, simple and intuitive test interpretation. But it’s not only advantageous from a psychological perspective. Increasing intensities are easier to interpret for the human eye, which is a performance related argument for a more sensitive analysis.

For these reasons, the following part of the article swaps the C and T lines according to the guidelines of current papers on SHERLOCK- and DETECTR-methods.

BTW: Determining nuclease activity with lateral flow and a reporter is not entirely new. This form of lateral flow-based readout was already used in 2015 in connection with prostate cancer diagnosis and correlating DNAse I activity. A dsDNA reporter is used here in combination with a selfmade teststrip. This LFD has only one test-line, which is analogous to the control-Line of the HybriDetect. A loss of signal correlated with an increasing DNase I activity. (10)

How to use the reporter to eliminate the Test-line?

The almost complete extinction of the T-line is the basis for an easy and intuitive interpretation of the test strips. In order to eliminate T-line intensity as much as possible, it is necessary to determine the required amount of reporter in the LFA. The goal is to find the perfect amount of reporter, that the majority of the gold conjugate is retained at the C-Line. Only few gold nanoparticles will be able to travel to the T-line, leading to a missing or very weak T-line. For an almost complete elimination of the T-Line a special amount of reporter is needed.

Figure 5. Impact of reporter amount on C- and T-line intensities. A relatively long ssDNA-reporter was used (length: 32 b, 5’ FITC and 3’ Biotin). 20 µL of diluted reporter was mixed in 80 µL Assay buffer in a 96-well plate. HybriDetect was placed contemporaneous into wells using the 12-Strip-Dip-Template. Results are read after 3 minutes.

We found that a range of 0.2 – 2.0 pmol per LFA is good for an almost complete T-line elimination. Comparable C- and T-line intensities are generated for 0.02 pmol and 100 pmol reporter per LFA, although the best result was achieved in between these two concentrations. This apparently surprising result can be explained by a typical immunoassay associated phenomenon which also occurs in lateral flow tests: the high dose hook effect.

CAUTION: too much reporter causes the High Dose Hook Effect

The high dose effect is a typical immunoassay related phenomenon. It occurs, if too many LFA-relevant labels (Biotin / FITC / FAM / DIG) are introduced into the LFD. The general mechanism is based on a limited number of label-specific binding sites in the lateral flow system. If the number of relevant labels exceeds the number of available binding sites in the lateral flow system, the overall number of signal-creating “molecular sandwiches” will be reduced. A decreasing C-line and increasing T-line intensity is the result of a significant reporter excess. This is an important and characteristic limitation for immunoassay formats like LFAs or ELISAs. The following figure illustrates the reporter-induced hook effect in the HybriDetect system.

Figure 6. Reporter induced Hook Effect in the HybriDetect system.

It is necessary to understand the hook effect, to avoid misinterpretation and handling errors. According to this effect it is possible to create identical signal intensities with different concentrations of the dual labeled reporter. Furthermore, it is possible to create false negative results especially for assays, which are designed according to the described label incorporation strategy. It is crucial to limit the number of relevant labels in the LFA.

Figure 7. Idealized C- and T-line intensity depending on the reporter concentration in the LFA to illustrate the reporter induced hook effect in the HybriDetect system.

In other experimental settings we tried to determine the maximum number of labels per LFA without running into the hook effect. The following table gives a brief overview of the discussed limitations.

Table 2. Overview of the maximum label amounts before running into the hook effect. T-Lines are defined according to the instruction for use, and not according to CRISPR/Cas-related HybriDetect usage.

Tips and tricks for sufficient T-Line Elimination

# Good range for reporter concentration: 0,1 – 2 pmol / LFA
# Too little amount of reporter and too much reporter will result in increasing T-Line intensity >> difficult to interpret
# CAUTION >> Too much reporter induces high dose hook effect
# Long reporter sequence can result in secondary structures, or reporter-reporter interaction >> possible influence on the detectability
# Pretest new reagents and new lots

Advanced Tips

The use of 1 or 2 pmol may not be the optimum for a quick and efficient measurement of nuclease activity. If the reporter concentration in the nuclease assay deviates from T-line elimination range, the T-line intensity will be too strong for an intuitive interpretation. But there are some valuable tricks to achieve the best possible readout.

SPIKE IN: additional non cleaved reporter can be added to the Assay Buffer. Generally, you can use ssDNA reporter and increase stability of spiked assay buffer independent of the nuclease assay type. This is an easy tool for test-line intensity finetuning, if the reporter amount is not sufficient for T-line elimination. Assay buffer modification is an underrated tool in HybriDetect associated analysis.

DILUTE / less SAMPLE: at the end it’s all about pretesting your test system in the LFA context. Try to be confident using less sample or dilute before LFA. Less can be more!

SAMPLE on CRP: Try to applicate your sample directly on the Conjugate Release Pad (max. 10 µL) before you dip the stick into the assay buffer (in addition to the reporter SPIKE IN). The potentially cleaved reporter will have an advantage to bind to the mobile antibodies. Sometimes this can be beneficial to achieve sensitive test results.

TIME: Analysis time is extremely important. Two – three minutes of Lateral Flow are most frequently used in the reporter degradation dependent methods until documentation. The contemporaneous analysis start is important and can be challenging with 10 or more strips. To achieve comparable results regards analysis time, try to implement the 12-Strip-Dip-Template (can be downloaded here) in your assay procedure.

References:

1. Myhrvold C et al., 2018. Field-deployable viral diagnostics using CRISPR-Cas13.
2. Abudayyeh OO et al., 2019. Nucleic Detection of Plant Genes Using CRISPR-Cas13.
3. Sullivan et al., 2019. Rapid, CRISPR-based, Field-Deployable Detection of White Spot Syndrome Virus in Shrimp.
4. Zhang et al., 2020. A protocol for detection of COVID-19 using CRISPR diagnostics.
5. Metsky et al., 2020. CRISPR-based surveillance for COVID-19 using genomically-comprehensive machine learning design.
6. Kaminsky et al., 2020. A CRISPR-based assay for the detection of opportunistic infections post-transplantation and for the monitoring of transplant rejection.
7. Broughton et al., 2020. CRISPR–Cas12-based detection of SARS-CoV-2.
8. Tsou et al., 2019. A CRISPR Test for Detection of Circulating Nuclei Acids.
9. Zhang Y. et al., 2020. Evaluation of CRISPR/Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice.
10. Zhang Y and Ying YY, 2015. Homogeneous Immunochemical Assay on the Lateral Flow Strip for Measurement of DNase I Activity.

The CRISPR/Cas-enzymes Cas12 or Cas13 are activated by binding to a specific target. When activated, the endonucleases are able to cleave nonspecific reporters.

A simple, instrument-free and portable way to readout the SHERLOCK reaction is using our LFA (lateral flow assay) HybriDetect.

The Way HybriDetect is used in the SHERLOCK Assay:

A reporter labeled with FITC on one side and Biotin on the other side has to be used. Upon activation of Cas12/Cas13 this reporter is cleaved. The biotin side of the reporter is captured by the lower band of the HybriDetect dipstick. Anti-FITC antibodies, which are labeled with gold nanoparticles, will bind on the other side of the Reporter (FITC). A dark purple line is formed at the Control band of the dipstick. Upon activation of Cas12/cas13 by binding the specific target sequence the reporter is cleaved. The gold NP-labeled antibodies, which are bound to the FITC side of the reporter will travel across the test band of the dipstick. Here they are bound by species specific antibodies and form a purple line at the 2nd position.

Reference:

Kellner M. J., Koob J. G., Gootenberg J. S., Abudayyeh O. O., Zhang F., SHERLOCK: nucleic acid detection with CRISPR nucleases. Nature Protocols (2019); V 14 (10, 2986-3012)

protein(s) to the viral genome. A characteristic feature of Cas proteins is the endonuclease activity, which causes the specific degradation of viral nucleic acids. These combined features of sequence specific recognition and cutting have been used for the development of genome editing tools. (3, 4)

Figure 1. The steps of CRISPR-mediated immunity. adapted from Molecular Cell 54, April 24, 2014. (5)
Importance of Diagnostic Alternatives in the Point-of-Care Field

There is a great need for diagnostic alternatives that are suitable for simple, fast, specific, sensitive, and inexpensive early detection of pathogens. Simple handling and the avoidance of expensive and complex devices are considered to be particularly important, especially for third world countries or regions with limited lab capacities. (6, 7)

This deficiency became very clear in the years 2014 to 2016 during the Ebola outbreak (8). Today we are experiencing an even more extreme situation. The Sars-CoV-2 pandemic affects the whole world and safe, scalable diagnostics is one of the most important issues today. As part of WHO’s response to the outbreak, the R&D Blueprint has been activated to accelerate diagnostics, vaccines and therapeutics for this novel coronavirus (9).
What Makes CRISPR Associated Enzymes Attractive for Diagnostic Purposes?

In recent years, CRISPR/Cas-based detection systems have increasingly come into focus as serious diagnostic alternatives. But why are some Cas proteins particularly suitable for molecular biosensing?
Within the Cas protein classification model, all diagnostically relevant Cas proteins originate from class II. These are multidomain proteins that are guided to the target nucleic acid by associated single stranded RNAs. Another essential feature of diagnostically relevant Cas proteins is a so-called collateral activity, which occurs after a target sequence has been successfully recognized. Single-stranded RNA-/ DNA-fragments are efficiently degraded in a short period of time due to this collateral activity. And this function makes these proteins such interesting tools in diagnostics. By using molecular reporters, the collateral activity can be used for signal generation in a diagnostic approach. Reporters are short DNA- or RNA-fragments that have defined labels at their ends. Reporter cleavage leads to the generation of a detectable signal.

CRISPR/Cas-dependent Reporter Cleavage via Collateral Activity

Different Cas proteins have special characteristics and differ in some relevant criteria such as: size, recognition of nucleic acid type, collateral degradation of ssDNA or ssRNA (10). Table 1 gives a brief overview of the three diagnostically relevant Cas proteins and their specific features.

Table 1. Characteristics of commonly used CRISPR-associated Proteins for diagnostic purpose

CRISPR/Cas-Systems and Lateral Flow Readout with HybriDetect

CRISPR/Cas-based detection methods can be combined with a simple Lateral Flow Readout. Therefore the universal lateral flow platform HybriDetect is the perfect tool for a sensitive, rapid, equipment-free and simple visualization of test results (11). The general mechanism is explained in the following Figure 3.

Figure 3. General mechanism of CRISPR/Cas-mediated detection of nucleic acids via HybriDetect Lateral Flow. (A) The presence of the genetic target leads to a positive test result. (B) The absence of the specific genetic target leads to a negative test result.

The interpretation of the HybriDetect dipstick is extremely simple. If the intensity of the T-line exceeds the T-line intensity of the negative control, the test is interpreted as positive. At the same time, the C-line intensity decreases in clear positive results. The simple and intuitive interpretation of the test strips is illustrated in the following figure. About 200 copies of artificial virus RNA can be clearly detected using the CRISPR/Cas-based detection method in combination with HybriDetect lateral flow assay.

Figure 4. Detection of synthetic ZIKA virus ssRNA using SHERLOCK with 1 hour of LwaCas13a reaction, followed by Lateral Flow with Milenia HybriDetect. (12)

CRISPR/Cas-detection methods are mostly combined with pre-amplification steps. Isothermal amplification such as LAMP or RPA are the most frequently used techniques. Combinations of these methods are named SHERLOCK, DETECTR or HOLMES. Recently, it has been proved that these CRISPR/Cas-methods are able to detect pathogenic viral genomes at attomolar levels in a simple, rapid and low-equip Point-of-Care-approach using the Milenia HybriDetect.

# Detection of Zika Virus , SHERLOCK_CRISPR/Cas13-System (12,13)
# Detection of Dengue Virus, SHERLOCK_CRISPR/Cas13-System (12)
# Detection of Human Papillomavirus (HPV) -16 and -18, CRISPR/Cas12a-System (14)
# Detection of SARS-CoV-2, CRISPR/Cas12a-System, CRISPR/Cas13-System (15,16)

References

Jinek M, Chylinski K, Fo nfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual‐RNA–guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096): 816‐821. DOI: 10.1126/science.1225829, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286148/pdf/nihms-995853.pdf

Wright AV, Nuñez JK, Doudna JA. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell. 2016;164(1‐2):29‐44. DOI: https://doi.org/10.1016/j.cell.2015.12.035

Singh V, Gohil N, Ramírez García R, Braddick D, Fofié CK. Recent advances in CRISPR‐Cas9 genome editing technology for biological and biomedical investigations. J Cell Biochem. 2018;119(1):81‐94. https://doi.org/10.1002/jcb.26165

Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA‐guided endonuclease of a class 2 CRISPR‐Cas system. Cell. 2015;163(3):759‐771. Doi: 10.1016/j.cell.2015.09.038.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4638220/pdf/nihms725840.pdf

Barrangou, R. and Marraffini, L. CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity (2014). Molecular Cell 54, 234-244. Original Image: http://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/

Zumla A, Al-Tawfiq J, Enne V, Kidd M, Drosten C, Breuer J, Muller M, Hui D, Maeurer M, Bates M, Mwaba P, Al-Hakeem R, Gray G, Gautret P, Al-Rabeeah A, Memish Z, Gant V. Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections-needs, advances, and future prospects. The Lancet Infectious Diseases. 2014 vol: 14 (11) pp: 1123-1135. Doi: 10.1016/S1473-3099(14)70827-8.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106435/pdf/main.pdf

Chen H, Liu K, Li Z, Wang P. Point of care testing for infectious diseases.Clinica Chimica Acta. 2019 vol: 493 pp: 138-147. Doi: 10.1016/j.cca.2019.03.008. https://doi.org/10.1016/j.cca.2019.03.008

Pollock NR, Wonderly B. Evaluating novel diagnostics in an outbreak setting: lessons learned from Ebola. J Clin Microbiol. 2017;55(5):1255‐1261. https://jcm.asm.org/content/jcm/55/5/1255.full.pdf

WHO, A research and development Blueprint for action to prevent epidemics. Source: https://www.who.int/blueprint/en/

Aman R, Mahas A, Mahfouz M. Nucleic Acid Detection Using CRISPR/Cas Bio-sensing Technologies. ACS Synthetic Biology. 2020. doi:10.1021/acssynbio.9b00507. https://pubs.acs.org/doi/10.1021/acssynbio.9b00507

James A, Todd S, Pollak N, Marsh G, Macdonald J.Ebolavirus diagnosis made simple, comparable and faster than molecular detection methods: Preparing for the future.Virology Journal. 2018 vol: 15 (1). https://doi.org/10.1186/s12985-018-0985-8.

Gootenberg J, Abudayyeh O, Kellner M, Joung J, Collins J, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science vol. 360, pp: 439-444 (2018). doi: 10.1126/science.aaq0179. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961727/pdf/nihms965860.pdf

Myhrvold C, Freije C, Gootenberg J, Abudayyeh O, Metsky H, Durbin A, Kellner M, Tan A, Paul L, Parham L, Garcia K, Barnes K, Chak B, Mondini A, Nogueira M, Isern S, Michael S, Lorenzana I, Yozwiak N, Macinnis B, Bosch I, Gehrke L, Zhang F, Sabeti P. Field-deployable viral diagnostics using CRISPR-Cas13. Science vol. 360, pp: 444-448 (2018). Doi: 10.1126/science.aas8836. https://science.sciencemag.org/content/360/6387/444/tab-pdf

Tsou J, Leng Q, Jiang F. A CRISPR Test for Detection of Circulating Nuclei Acids. Translational Oncology. 2019 vol: 12 (12) pp: 1566-1573. doi: 10.1016/j.tranon.2019.08.011 https://www.sciencedirect.com/science/article/pii/S1936523319304140?via%3Dihub

Broughton J, Deng X, Yu G, Fasching C, Streithorst J, Granados A, Sotomayor-Gonzalez A, Gopez A, Hsu E, Gu W, Miller S, Pan C, Wadford D, Chen J, Chiu C, Chiu C.Rapid Detection of 2019 Novel Coronavirus SARS-CoV-2 Using a CRISPR-based DETECTR Lateral Flow Assay. PREPRINT server: bioRxiv. Doi: 10.1101/2020.03.06.20032334. https://www.medrxiv.org/content/10.1101/2020.03.06.20032334v2

Metsky H, Freije C, Kosoko-Thoroddsen T, Sabeti P, Myhrvold C. CRISPR-based surveillance for COVID-19 using genomically-comprehensive machine learning design. PREPRINT server: bioRxiv. 2020 pp: 2020.02.26.967026. doi: 10.1101/2020.02.26.967026. https://www.biorxiv.org/content/10.1101/2020.02.26.967026v2.full.pdf

gene amplification products resulting from PCR, LAMP or RPA. The underlying genetic information can be detected regardless which kind of organism need to be detected. Finally, labeled primers must be introduced in the amplification step, so that the resulting fragments are labeled and can be detected by the HybriDetect dipstick.

Results can be reported within 5 minutes after lines become visible on the test strip.
If you want to learn more about HybriDetect, for example information on the limit of detection, see our other blog articles.

COVID-19 Detection Methods

At the moment, the standard COVID 19 test is a qRT-PCR (quantitative real time polymerase chain reaction). Obviously, there are several reasons why qRT-PCR test cannot be used as a POCT (point of care test). First of all, it takes several hours to do a qPCR test, second you need highly trained personnel and third special equipment is essential. In the current situation, with the entire world dealing with the COVID-19 outbreak, it is of great interest to develop rapid tests for POC testing of the new corona virus to help people in sparsely populated areas where laboratories are difficult to reach.

Since the outbreak of COVID-19 at the end of 2019 several tests for SARS-CoV-2 detection have been developed and approved. But most of them show several disadvantages:

# Expensive laboratory equipment is needed
# High level technical expertise is required
# Access to reagents is difficult
# Expensive

With the currently available rapid Antigen tests, a result is available after 15 minutes outside the lab. But the Antigen tests are detecting viral proteins, and are therefore less sensitive as a test on DNA level.

Due to these reasons, several groups are working on alternative tests for the quick, easy and low cost detection of SARS-CoV-2 with very little equipment needed.

Why do we need more rapid tests?

The head of the WHO Tedros Adhanom Ghebreyesus already told on March 16th, that there has not been an urgent enough escalation in testing. His key message on this day was: test, test, test.

The most important point is to detect local outbreaks as soon as possible and control the spread of the COVID-19 virus through quarantine measures. The effect of the high test capacities could be seen in South Korea. They rely on early PCR tests to record lots of cases, even mild and symptom free ones. Tests are offered in drive troughs and walk troughs. Consequently, the number of infections in South Korea stayed relatively low.

At a school in Mecklenburg-West Pomerania, in Germany all teachers and students, even their parents are offered free tests to identify and contain infections early. The tests are voluntary and offered twice a week. Mecklenburg-West Pomerania is planning to do the COVID-19 tests in every school, to get back to “normal life conditions” quickly.

These two examples show the high need for frequent testing. Obviously, obtaining fast test results is one of the biggest advantages of point-of-care compatible rapid tests. Generating quick test results without the need of sending the samples to an external laboratory allows quick reaction and can help to control local outbreaks.

Here we give a short overview of the latest publications dealing with the rapid detection of

COVID-19 combined with the HybriDetect Lateral Flow Method for visualization.

COVID 19 testing and CRISPR

Promising testing methods, which are based on isothermal amplification in combination with a CRISPR-mediated detection (SHERLOCK (1), DETECTR (2)) have been developed.

SHERLOCK

The group of the inventor of the SHERLOCK method Feng Zhang and collegues, based in the United States, described a method for COVID-19 detection using CRISPR. The scientists were able to detect synthetic COVID-19 virus RNA fragments between 20 and 200 aM (10-100 copies per µl of input). They used purified RNA as Input for an RT-RPA before the Cas13 assay. The whole method takes less than one hour.

The following picture shows an example of the COVID-19 detection using our HybriDetect.

Fig. 1: Covid-19 testing. Detection of the Covid-19 S gene with HybriDetect Strips. First four dipsticks show positive result.

For more information see the original paper:

https://www.broadinstitute.org/files/publications/special/COVID-19%20detection%20(updated).pdf

Zhang et al give a precise description, how the SHERLOCK technique is done in nature protocol:

http://dx.doi.org/10.1038/s41596-019-0210-2

STOPCovid – improved SHERLOCK method

Due to the fact, that the method described above needs two reaction steps (isothermal amplification and CRISPR-reaction) it is still complex and cross-contamination in an uncontrolled environment can happen quickly. For these reasons, Zhang and colleagues recently published an update (preprint, not peer reviewed) on the SHERLOCK method, which can be performed in one step (3) and is a promising rapid COVID 19 detection method.

The SHERLOCK method was continously improved in the last few years to make specific POC tests possible:

2016: Detection of Cas13 by Zhang lab
2018: Improvement of the Cas enzymes Cas12b and Cas13 (SHERLOCKv2)
2019: First paper on the specific detection of DNA and RNA through CRISPR-Cas from clinical relevant samples. Step-by-step instructions with RPA
2020: STOPCovid: One Pot reaction combines LAMP and SHERLOCK to detect Sars-CoV-2

The so-called STOP (SHERLOCK Testing in One Pot) method is an improvement of the previous SHERLOCK application. This test allows a turnaround time of an hour from sampling to the report of the resuls. The very simple handling underlines the potential as a point-of-care test (POCT) for COVID 19 testing.

General information about STOP:

~Results in 40 to 70 minutes
~Detection of 100 copies of viral genome
~Saliva or nasopharyngeal swabs as input
~Specific detection
~Single temperature, one fluid handling step, visual readout
~Nearly no equipment

Fig. 2 Equipment needed for STOP. Beside a simple heat block, some tubes, pipettes and tips, HybriDetect lateral flow strips need to be used for COVID19 testing.

One pot reaction in three simple steps

Step 1: 10 mins at 22°C or at 60°C → lysis of virus-containing patient sample (add Proteinase K inhibitor) or 5 mins at 95°C

Step 2: 1 hr at 60°C → detection of viral RNA using STOPCovid reaction

Step 3: 2 mins at 22°C → visual read out of the detection results by eye using HybriDetect

Fig. 3: STOPCovid in five steps – from sample to result.

If you want to learn more about the lateral flow readout of HybriDetect in combination with CRISPR, read our related article: Lateral Flow Readout for CRISPR/Cas-based detection strategies

Combining RT-LAMP, Cas12b and HybriDetect

The authors used a RT-LAMP (loop-mediated isothermal amplification) for RNA-transcription followed by simple isothermal DNA-amplification, which works at 55-65°C. For this reason they needed a thermostable Cas-Protein, which is active at 60°C. They found Cas12b from Alicyclobacillus acidiphilus (Aap) to be stable at this temperature. Out of 29 primer sets for detecting SARS-CoV-2 the scientists found LAMP-compatible primer sets. The next step was to find the right guide RNA (gRNA), which was figured out comparing 18 gRNAs.

During another optimization it was discovered that the addition of taurin resulted in an improvement of the reaction kinetics. After RT-LAMP and CRISPR-reaction (Step 3 in Figure 2) HybriDetect can be dipped directly into the reaction tube at room temperature and visual read out can be done after 2 minutes.

The Authors provide all the information needed to do the STOPCovid test in their paper: LAMP Primers, gRNA sequence, Cas12b-Protein Sequence, etc.

Validation of STOPCovid

The method was validated on 17 patient samples. It could detect 12 positive and 5 negative samples, 2 of 3 replicates were scored positive in infected patients. STOPCovid was able to detect all positive and all negative samples (Fig. 3) compared to the gold standard method RT-qPCR.

Fig. 4: Validation of STOPCovid. Results for 17 unique SARS-CoV-2 nasopharyngeal swab samples. Quantification of the band intensity ratio of lateral flow results.

STOPCovid.v2

The inventors of STOPCovid are constantly improving the Covid-19 detection method for rapid testing. In theire latest update on the method they described a magnetic bead purification method. With this approach, they could reduce the sample extraction time to 15 minutes. (Fig. 5)

The researchers compared the STOPCovid.v2 with the gold standard RT-qPCR. The result was a reduction of viral RNA that was 600 times of the input needed for the RT-qPCR. The STOPCovid.v2 has a limit of detection (LOD) similar to a cycle-theshold (Ct) value of 40.3. StopCovid.v2 was tested on 202 SARS-CoV-2 positive and 200 SARS-CoV-2 negative samples. The test result showed a sensitivity of 93.1% and a specificity of 98.5%. The positive samples were detected in 15 to 45 minutes.

Read the original article here: https://www.nejm.org/doi/full/10.1056/NEJMc2026172?query=featured_home
What is the clue about the updated version of SHERLOCK?

Taken together the striking advantage of this method is its simplicity. The 3 steps, shown above are very easy to do, even for untrained personnel. The authors even showed that saliva samples worked as input for the reaction. This makes the method performable for lay users. Another advantage is the low-tech equipment approach, which has a major impact on the POC-compatibility.

Scientists who are interested in testing the protocol can get more information and test kits at STOPCovid.science.

The protocol is not authorized by the FDA (food and drug administration), it is not for clinical purposes.

Read the original article here: https://www.medrxiv.org/content/10.1101/2020.05.04.20091231v1.full.pdf

DETECTR

Broughton et al. already published a paper, how to detect SARS_CoV-2 using LAMP, Cas12 and our HybriDetect within 30 minutes. They are using isothermal pre-amplification with primers published by the WHO and CDC. As the authors mentioned in the paper, this application would be a helpful tool for POCT testing in emergency departments, airports etc..

Here you can find a video from the McGovern Institute describing the SHERLOCK technique in combination with the HybriDetect in General

In summary, rapid testing is being expanded to stop the spread of Covid 19. Scientists in the United States and India (read more about the Feluda Test) developed new tests on Genome level, which are nearly as fast as rapid antigen tests, but more sensitive. With these new approaches, departments of health could be relieved and the public health improved.

References

1. Zhang et al., 2020. A protocol for detection of COVID-19 using CRISPR diagnostics.
2. Broughton et al., 2020. CRISPR–Cas12-based detection of SARS-CoV-2.
3. Joung et al., 2020. Point-of-care testing for COVID-19 using SHERLOCK diagnostics