In order to monitor and contain the spread of SARS-CoV-2 it is necessary to test large numbers of people on a regular basis in decentralized settings.
Researchers of the Max Planck Institute for Evolutionary Anthropology and the Hospital St. Georg in Leipzig, Germany, have developed improved protocols for the detection of SARS-CoV-2. The method can detect a positive sample in a pool with 25 uninfected samples in less than one hour.
Quantitative real-time polymerase chain reaction (qPCR) is the most widely used diagnostic method to detect RNA viruses such as SARS-CoV-2. However, it requires expensive laboratory equipment and global shortages of reagents for RNA purification has increased the need to find simple but reliable alternatives. One alternative to the qPCR technology is RT-LAMP (reverse transcription loop-mediated isothermal amplification).
This test amplifies the desired target sequences of the virus at a constant temperature, using minimal equipment compared to qPCR. In 2020, it was adapted to the detection of SARS-CoV-2. It was also shown that instead of a swab, which many people find unpleasant, it can be performed on gargle lavage samples.
First author Lukas Bokelmann and colleagues have now developed an improved colorimetric RT-LAMP assay, called Cap-iLAMP (capture and improved loop-mediated isothermal amplification), which extracts and concentrates viral RNA from a pool of gargle lavage samples.
After a short incubation, the test result – orange/red for negative, bright yellow for positive – can be interpreted visually or by using a freely available smartphone app.
The improved testing method outperforms previous similar methods. “Cap-iLAMP drastically reduces false positives and single infected samples can be detected in a pool among 25 uninfected samples, thus reducing the technical cost per test to only about 1 Euro per individual,” says senior author Stephan Riesenberg, a researcher at the Max Planck Institute for Evolutionary Anthropology.
“Our method overcomes problems associated with standard RT-LAMP and could also be applied to numerous other pathogens.”
Reverse transcription followed by quantitative PCR (RT-qPCR) is the most widely used method to detect RNA viruses such as SARS-CoV-2. However, its need for trained personnel and expensive instrumentation and global shortages of resources for RNA purification has spurred search for viable alternatives. Loop-mediated isothermal amplification (LAMP) can rapidly amplify small target nucleic acid sequences under isothermal conditions (Notomi et al. 2000) and has been applied for molecular diagnostics (Wong et al. 2018).
The reaction requires four to six primers and produces concatemers of double-stranded amplification products. These can be detected directly, using intercalating dyes (e.g. SYBR green, SYTO-dyes), its reaction with triphenylmethane dye precursors and acid hydrolysis (Miyamoto et al. 2015; Trinh and Lee 2019), or using cleavage with CRISPR enzymes coupled with lateral flow color detection of the cleavage product (Broughton et al. 2020).
However, these methods require opening of the tube after the reaction, thus posing the threat of cross-contaminating future reactions with the amplified product. Amplification can also be detected indirectly by hydroxynaptholblue or phenol red based detection of the release of protons and/or pyrophosphate generated during DNA synthesis (Tanner et al. 2015).
Recently, a number of studies explored ways to detect SARS-CoV-2 RNA using RT-LAMP (Lamb et al. 2020; Yang et al. 2020; Zhang et al. 2020) but they required time consuming RNA isolation steps before the reaction. There have also been attempts to add sample directly into the reaction without prior purification (Buck et al. 2020; Dao Thi et al. 2020).
However, it was noted that the pH of nasopharyngeal swab samples often varies and can adversely affect readouts.
Here we describe a method to detect SARS-CoV-2 RNA of a single infected individual within a bulk sample comprised of up to 26 individual patient samples by combining a hybridization- capture-based RNA extraction approach with smartphone app-assisted colorimetric detection of RT-LAMP products, a procedure that can be performed in less than one hour (Figure 1A).
We compared the sensitivity of the method to standard extraction RT-qPCR protocols in a diagnostic lab and validate its performance on 287 gargle lavage samples from a hospital, a nursing home previously affected by COVID-19, and round robin samples from a reference institution of the German Medical Association.
Development of Cap-iLAMP
We evaluated three published RT-LAMP primer combinations targeting either the Orf1a gene or the N gene of the SARS-CoV-2 genome (Lamb et al. 2020; Zhang et al. 2020) using a dilution series of synthetic viral RNA (Twist Biosciences, San Francisco, CA, USA) and chose the two most sensitive primer sets (CV1-6 and CV15-20, Supp. Table 1) to detect the Orf1a gene and the N gene for further testing (Figure 1B, Supp. Figure 1, Supp. Table 1).
Using both primer combinations, we detected 500 synthetic viral RNA copies after 25-30 minutes incubation at 65°C as measured by fluorescence real time RT-LAMP (Supp. Figure 1A and C). Combining the primers for the Orf1a gene and the N gene did not increase sensitivity (Supp. Figure 1D).
Amplification in LAMP reactions is often detected colorimetrically by a pH sensitive dye that changes color when extensive DNA synthesis lowers the pH of the reaction (Tanner et al. 2015). As noted in a previous study (Dao Thi et al. 2020), biological samples such as nasopharyngeal swab eluates may change the pH when added to the LAMP reaction directly, leading to false positive results.
We found that nasopharyngeal swab eluates tend to be more acidic than gargle lavage samples and that adding gargle lavage directly to a LAMP reaction at a final concentration of 5% leads to false positive results in 4.7% of cases even before the isothermal incubation (Supp. Figure 2A and B).
It would therefore be preferable to detect the amplification product directly rather than indirectly by a pH change. To achieve this, we deposited a drop of 0.5 microliters of a 10,000x concentrated solution of the dye SYBR Green I to the cap of the tube before the reaction is initiated. Shaking the tube after the isothermal amplification dissolves SYBR Green I, stops the reaction and allows visual detection of SARS-CoV-2 via a color change from orange/red to intense yellow (Figure 1C).
The color of the reaction can be objectively quantified as a single numerical hue value, that is insensitive to light intensity changes and can be derived from the red, green, blue (RGB) color model (Cappi et al. 2015; Yin et al. 2019), by using freely available ‘camera color picker’ smartphone apps.
We used the ‘Palette Cam’ app (Alexander Mathers, App Store) for extracting RGB values before conversion to hue. An additional advantage is that this detection strategy allows us to include acidifying enhancing enzymes in the LAMP reaction, namely Tte UvrD helicase, which prevents unspecific late amplification of artefacts induced by primer interactions (Supp. Figure 3A and B) and thermostable inorganic pyrophosphatase which increases reaction speed (Miyamoto et al. 2015).
However, addition of patient gargle lavage directly to this improved LAMP (iLAMP) reaction promoted false positives (13.5 %) (Supp. Figure 4A). Presumably, this unspecific amplification is due to DNA from the oral microbiome, food or host cells as it can be prevented by prior λ exonuclease treatment that preferentially digests 5’-phosphorylated DNA leaving non- phosphorylated primers and iLAMP product intact (Supp. Figure 4B).
Because, gargle lavages from known infected patients did result in false negatives for all seven samples we employed an initial Quick extract (Lucigen, Middleton, WI, USA) lysis (Ladha et al. 2020) to release more viral RNA from the capsid. Even when these optimized reaction conditions were used, reactions resulted in one false negative out of the seven gargle lavages from infected patients and the single SARS-CoV-2 negative gargle lavage sample was false positive (Supp. Figure 4B).
We therefore employed a rapid (15min) bead-capture enrichment purification akin to mRNA isolation, using two oligonucleotides flanking the RT-LAMP target sites (Figure 1B, Supp. Table 1) immobilized on paramagnetic beads. This step eliminates non-target nucleic acids and other unwanted components in the biological samples but also concentrates the viral RNA.
Comparing the Ct-values of RT-qPCR targeting the E gene (Corman et al. 2020) after silica-based RNA-extraction with hybridization capture targeting the E gene for the same volume of gargle lavage suggests a capture efficiency of roughly 5% (Figure 1D).
To allow comparison to RT-qPCR we captured viral RNA with a biotinylated probe for the E gene and not for the Orf1a and N gene as used for RT-iLAMP. When RNA is concentrated from 500µl gargle lavage to 25µl final volume and 10µl input volume is used in iLAMP, this results in a detection limit of 5-25 viral genome copies per µl of sample before capture.
The reagents required for the iLAMP reaction can be pre-mixed and freeze-thawed at least twice. Cap-iLAMP could be performed at point-of-care as no bulky equipment and only pipettes, a thermoblock, and a magnetic rack are needed (Figure 1E).
Detection of SARS-CoV-2 in patient gargle lavage samples
We used Cap-iLAMP to test 287 gargle lavage samples from hospital patients and elderly nursing home inhabitants and employees either individually or in pools. These samples had previously been tested by a RT-qPCR assay targeting the SARS-CoV-2 E gene (Maricic et al. 2020) and showed either no amplification or Ct values ranging from 24.6 to 32.7 (Figure 2A).
Cap-iLAMP targeting the SARS-CoV-2 Orf1a gene or the N gene results in an orange/red and intense yellow color for SARS-CoV-2 RNA negative and positive samples, respectively (Figure 2B). Of the 12 samples which we tested individually (Figure 2C), six had been previously tested positive in the RT-qPCR assay. Scoring the color of the iLAMP reactions using a smartphone ‘camera color picker’ app shows that hues for SARS-CoV-2 negative and positive samples are clearly separated, below 26° for the former and above 37° for the latter, respectively.
All six negative samples were correctly identified as negative in the Cap-iLAMP assays targeting the Orf1a and the N gene. Of the six samples that were SARS-CoV-2 positive in the RT-qPCR assay, four were positive in the Cap-iLAMP assay while the remaining two were false negative.
When one twentieth of input volume were used they were positive, suggesting that some residual inhibition originating either from the biological sample or from lysis/binding buffer carryover exists in these extracts.
To investigate whether it is possible to detect single infected individuals in pools of gargle lavage samples, we created eleven pools of 25 patient samples each, all of which had been tested negative in RT-qPCR assay and in the Cap-iLAMP assays for the Orf1a and the N gene (Figure 2D).
In order to determine if components of the pooled gargle lavage still inhibit the RT-LAMP reaction after capture, we took subsamples of the 10 negative pools and added 1000 copies/µl of artificial viral RNA before Cap-iLAMP. All 10 pools were positive in both Cap- iLAMP assays, showing that there was no substantial inhibition in these extracts after capture.
To investigate whether it is possible to detect a single infectious individual within a pool, we added different single positive patient samples (Ct < 26) to three pools of healthy individuals so that 1/26 (3.8%) of the final volume was composed of the infected sample. In all three cases, the Cap-iLAMP assays targeting the Orf1a and the N gene were positive, demonstrating that a single infectious individual can be detected in a pool of 26 samples.
The two SARS-CoV-2 positive samples previously found to be inhibited when tested individually (Figure 2C) were correctly identified as positive when pooled (Figure 2D), potentially because inhibition is rare and is diluted out in the pool. An additional wash step after hybridization capture should thus be applied for individual sample diagnostic testing.
All tested gargle lavages from single healthy individuals (n=6) and 11 pools of 25 healthy individuals (n=275) correctly tested negative for both the Orf1a gene and N gene in the Cap- iLAMP assay (Figure 2C and E), indicating that false positive results which were sometimes observed when samples are added directly into the iLAMP reaction (Supp. Figure 4A) are effectively prevented by the hybridization capture extraction.
Detection of SARS-CoV-2 in heat-inactivated cell-culture supernatants
Finally, we validated Cap-iLAMP against a set of seven reference samples of heat-inactivated cell-culture supernatants provided by the “INSTAND” reference institution of the German Medical Association (Duesseldorf, Germany) for round robin testing. These contained various amounts of SARS-CoV-2 (ring 59, 61, 63 and 64), different coronaviruses (ring 60 HCoV OC43 and ring 65 HCoV 229E) or no virus (ring 62) and had previously been tested via RT-qPCR (Maricic et al. 2020) (Figure 3A).
To prevent sporadic inhibition as observed for some gargle lavage samples, the first wash step after capture hybridization was performed twice. As shown in Figure 3B, all four SARS-CoV-2-containing samples were correctly and consistently identified by both the Orf1a and N gene assays while all samples devoid of virus or containing a different coronavirus were correctly classified as being negative.
reference link:: https://doi.org/10.1101/2020.08.04.20168617
More information: Lukas Bokelmann et al. Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP, Nature Communications, 5 March 2021, DOI: 10.1038/s41467-021-21627-0