key: cord-0963946-gtvc4c0p
authors: Haq, Farhan; Sharif, Salmaan; Khurshid, Adnan; Shabbir, Imran; Salman, Muhammad; Badar, Nazish; Ikram, Aamir; Ahad, Abdul; Malik, Muhammad Faraz Arshad
title: Development Optimization and Validation of RT-LAMP based COVID-19 Facility in Pakistan
date: 2020-05-29
journal: bioRxiv
DOI: 10.1101/2020.05.29.124123
sha: a2bd62aa39c7ca10b49e31bb221e06f79c4e0837
doc_id: 963946
cord_uid: gtvc4c0p

The pandemic SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) has created a widespread panic across the globe especially in the developing countries like Pakistan. The lack of resources and technical staff are causing havoc challenges in the detection and prevention of this global outbreak. Therefore, a less expensive and massive screening of suspected individuals for COVID-19 is required. In this study, a user-friendly technique of reverse transcription-loop mediated isothermal amplification (RT-LAMP) was designed and validated to suggest a potential RT-qPCR alternate for rapid testing of COVID-19 suspected individuals. A total of 12 COVID-19 negative and 72 COVID-19 suspected individuals were analyzed. Both RT-qPCR and RT-LAMP assays were performed for all the individuals using open reading frame (ORF 1ab), nucleoprotein (N) and Spike (S) genes. All 12 specimens which were negative using RT-qPCR were also found negative using RT-LAMP assay. Overall 62 out of 72 positive samples (detected using RT-qPCR) were found COVID-19 positive using RT-LAMP assay. Interestingly all samples (45) having Ct values less than 30 showed 100% sensitivity. However, samples with weaker Ct values (i.e., => 35) showed 54% concordance, suggesting potential false negatives or false positives in RT-LAMP or RT-qPCR results, respectively. Overall comparative assessment showed that RT-LAMP assay showed strong sensitivity and specificity and can be used as an alternative strategy for rapid COVID-19 testing. Hence, based on fast processing time, minimal risk of specimens transfer and utilizing available resources, LAMP based detection of COVID-19 is strongly advocated especially for developing countries.

Global outbreak of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) is creating a havoc challenge especially in developing countries like Pakistan 1,2 . Being 5 th populated country, sharing borders with two adversely affected countries (Iran and China), a massive screening of suspected individuals for COVID-19 is required 3 . Based on variable clinical symptoms ranging from mild flu to life threatening shortage of breath signs it is pertinent to establish an efficient diagnostics tool for earliest detection SARS-CoV-2. So far all commercially COVID-19 tests are relying on two strategies. In the first strategy, variety of immunological assays are developed responsible for detection of antibodies produced by individuals as a result of current or past exposure to the virus 4 . The second strategy focusses on SARS-CoV-2 viral RNA detection using polymerase chain reaction (PCR) 5 . Additionally, loop mediated isothermal amplification-based techniques are in process 6 . All these strategies have preferential advantages depending upon the objectives of the screening. As immune assays help to identify individuals retaining developed antibodies against virus and could be potential convalescent plasma donors. Genome based testing facilities help in diagnosis of SARS-CoV-2-infected individuals.

As per National Command and Operation Control (https://ncoc.gov.pk/), there are 94 laboratories offering detection of COVID-19 in Pakistan. All these labs are using real time PCR techniques (RT-qPCR) for detection of SAR-Cov-19. Despite of technique's robustness, challenges related to increasing number of affected cases in both urban and rural areas, direly require fast and less expensive alternate techniques. Since installation of expensive instrument, prolong reaction time (~3hr), cost of reagents and trained staff availability in an emerging outbreak (COVID-19) are debilitating its effective utilization across Pakistan.

Here, an alternate user-friendly technique of reverse transcription-loop mediated isothermal amplification (RT-LAMP) was designed and validated against COVID-19. Earlier, LAMP assay was used for detection of Hepatitis B virus (HBV) 7 . Briefly, Bacillus stearothermophilus (Bst polymerase) along with set of 4 primers (2 inner and 2 outer primers) were used in the assay. Reaction began with the binding of the inner primers containing sequences of sense and anti-sense strands of DNA. Here, Bst DNA polymerase retain 5'→3' polymerase activity while lacking 5'→3' exonuclease activity for 1 hr at 65°C.

Addition of reverse transcriptase along with Bst polymerase in LAMP assay can also enhance its utilization for rapid detection of RNA based viruses. In light of these modifications, LAMP technology was efficiently been advocated by World Health Organisation (WHO) under ASSURED (Affordable, Sensitive, Specific, Userfriendly, Rapid and Robust, Equipment free, Deliverable to end users) criteria 8 .

National Institute of Health (NIH) is a core national referral resource Institute responsible for accreditation and testing of COVID-19 screening facilities. For the present study, 72 COVID-19 suspected individuals were enrolled at NIH. These individuals were categorized into different groups based on the Ct values retrieved from RT-qPCR.

Nasopharyngeal specimens from these individuals were collected at designated specimen collection facility. After immersing these specimens in virus transfer buffer, these tubes were processed further for RNA isolation using commercially available kit. TANBead Nucleic Acid Kit was used for viral RNA isolation by using TANBead Nucleic Acid Extractor (model SLA-16/32) as per manufacturer instructions. Briefly, this kit used silicon dioxide layer coated magnetic beads for isolation of nucleic acid in 40 min.

Primers targeting open reading frame (ORF 1ab), nucleoprotein (N) and Spike (S) genes were designed using Primer Explorer V5 software. As per guidelines of LAMP assay, these panel of primers were specifically designed against a predefined genomic region of 2019-nCoV (genome Reference Sequence: NC_045512.2). Primer sequences will be provided on request.

WarmStart Colorimetric LAMP 2X Master Mix (New England Biolabs) vials were used for RT-LAMP assay. Three separate primer mix for each gene were prepared as working stock vials for the reaction. Reaction tube for each specimen containing 2x master mix (12.5 µl), 10X primer mix (1X; 2.5 µl), RNA (2 µl) and water (8 µl) were left at thermomixer® (Eppendorf) for incubation. Efforts were made to set forth best optimum temperature ranging from 62C° -65C° with time ranging from 45 min-75min for better yield.

Being a colorimetric LAMP assay, change in color of reaction mix from pink to yellow color was set as indicator of presence of virus. Since generation of concatemers result in lowering of pH from basic to acidic state. Hence, both amplification and detection of respective concatemers were completed in a single step. In addition, samples were also validated using gel electrophoresis results.

All aforementioned specimens (72) were analyzed using available COVID-19 diagnostic kits. RNA sample of these respective specimens were processed for diagnosis using RT-qPCR kit as per manufacturer instructions. Hence RT-LAMP findings were also cross checked with available gold standard testing facility of RT-qPCR.

Majority of these individuals were male expatriate Pakistani returning home with median age of 38Yrs. All 12 specimens which were negative using RT-qPCR were also found negative using RT-LAMP assay.

Overall 62 out of 72 positive samples (detected using RT-qPCR) were found COVID-19 positive using RT-LAMP assay. After extensive optimization, RT-LAMP reaction optimum conditions for all three genes were set at 65C° for 1hr duration. COVID-19 positive samples showed pronounced change in coloration from pink to yellow color against all three respective genes (Figure 1 ). 

Overall comparative assessment showed that RT-LAMP assay showed strong sensitivity and specificity and can be used as alternate strategy for rapid COVID-19 testing. Earlier, LAMP assay been widely used for screening of food pathogens 9 , detection of avian influenza virus 10 and gastric cancer metastasis 11 . Interestingly, several modifications towards earliest identification of concatemers also improve and largely reduced assay dependency on expensive instruments. Conventionally, LAMP products could also be detected by using agarose gel electrophoreses after 1hr incubation at high temperature 7 . Later on, formation of white precipitate (magnesium pyrophosphate) in the reaction tube may also traced either by naked eye or spectrophotometry 12 

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In Vitro Diagnostic Assays for COVID-19: Recent Advances and Emerging Trends

Rapid Detection of COVID-19 Coronavirus Using a Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) Diagnostic Platform

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A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity-Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

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We are grateful to National Institute of Health, COMSATS University Islamabad and Higher Education for providing needful resource facilitation, training and funding resource. All authors declare no conflict of interest.

All authors have seen and approved the manuscript, and that it hasn't been accepted or published elsewhere.