Month: February 2021

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