Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: a reliable, faster and economical method

Gupta, Ekta; Padhi, Abhishek; Khodare, Arvind; Aggarwal, Reshu; Ramachandran, Krithiga; Mehta, Vibha; Kilikdar, Mousumi; Dubey, Shantanu; Kumar, Guresh; Sarin, Shiv Kumar

doi:10.1101/2020.04.25.20079095

Cited by 9 publications

(6 citation statements)

References 11 publications

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“…11 Another three studies performed group testing by taking relatively small pool sizes of 5 to 10 samples. [12][13][14] Abdalhamid et al observed that positive samples (detected by individual testing) remain positive even if pooled with 4 negative samples. They created 21 pools with each pool containing 5 samples (1 known positive + 4 known negative).…”

Section: Optimal Pool Sizementioning

confidence: 99%

“…Thirty four pools out of 35, showed concordance with individual testing, that is, 95.4% pools correctly identified the positive samples. 14 Two simulation studies parameterized various pool sizes and multistage testing schemes and compared their efficiency at different prevalence rates. 15, 16 Eberhardt et al found that three-stage schemes performed optimally for prevalence rates up to 12%, and that initial pool sizes of 16 samples were best for prevalence rates up to 3.5% and pools of 9 samples for rates between 3.5% and 12% (P9S3, improvement factor 3.8 to 1.5).…”

Section: Optimal Pool Sizementioning

confidence: 99%

“…All the included studies observed significant reduction in the number of total tests, depending on pool size and prevalence. [9][10][11][12][13][14][15][16][17] Abdalhamid et al observed expected number of tests per individual to be < 0.44 with optimal pool size of 5 and prevalence rate at 5% (►Table 2). 12 Sinnott-Armstrong et al also found maximum reduction in tests with 384-well plates, provided the prevalence is low (< 2%).…”

Section: Reduction In Number Of Reactionsmentioning

confidence: 99%

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Effectiveness of Sample Pooling Strategies for SARS-CoV-2 Mass Screening by RT-PCR: A Scoping Review

Deka

Kalita

2020

J Lab Physicians

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The ongoing COVID-19 pandemic has hugely impacted the economy of many countries, and there is an acute shortage of diagnostic resources. With the exponential increase in the number of cases and necessity to screen large number of people, there is a steep increase in the demand for diagnostic kits. Pooled-sample testing is a promising strategy to screen a large population rapidly with limited resources. The aim of this work was to compile a cohesive literature review of the effectiveness and accuracy of pooled-sample testing in the detection of SARS-CoV-2 and critically analyze its limitations. Medline, Google Scholar, Embase, and preprint servers (e.g., bioRxiv) were searched for literature on pooled testing for diagnosis of COVID-19, and out of initial 60 articles/reports, nine original articles were retained. Optimal pool size (number of samples in a pool) seemed to be dependent on factors like prevalence or rate of positivity in community. In low-prevalence localities pool size of around 30 seemed to be effective as observed by some authors. All the researchers had found significant reduction in number of tests (depending on pool size, stages, and pooling design), leading to conservation of resources. Pooling can be done with extracted RNA eluate or directly with patient’s sample before extraction. This leads to further reduction in consumables, time and manpower. Risk of false negativity in samples with high-threshold cycle (i.e., low-viral load) value was a concern. Some researchers suggest adding few additional cycles to lower the chances of missing positive cases with low-Ct value. Lower limit of detection (LoD) of RT-PCR kits, that is, sensitivity of kits was another factor to consider. Thus, in a country like India, given the economic benefit and scarcity of resources, pooling strategy can be very effective, especially in low-prevalence areas and in low-risk contacts.

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Section: Optimal Pool Sizementioning

confidence: 99%

Section: Optimal Pool Sizementioning

confidence: 99%

Section: Reduction In Number Of Reactionsmentioning

confidence: 99%

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Effectiveness of Sample Pooling Strategies for SARS-CoV-2 Mass Screening by RT-PCR: A Scoping Review

Deka

Kalita

2020

J Lab Physicians

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“…The pooling method was validated for nCov tests by many laboratories [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. However, the following questions remained open, which we address in this paper.…”

Section: Introductionmentioning

confidence: 99%

Pooling of coronavirus tests under unknown prevalence

Pikovski

2020

Epidemiol. Infect.

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Diagnostic testing for the novel coronavirus is an important tool to fight the coronavirus disease (Covid-19) pandemic. However, testing capacities are limited. A modified testing protocol, whereby a number of probes are ‘pooled’ (i.e. grouped), is known to increase the capacity for testing. Here, we model pooled testing with a double-average model, which we think to be close to reality for Covid-19 testing. The optimal pool size and the effect of test errors are considered. The results show that the best pool size is three to five, under reasonable assumptions. Pool testing even reduces the number of false positives in the absence of dilution effects.

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“…This compromise in cycles was present in both probe-and dye-based assays. However, an increase of CT values from 33,1 to 35,6 cycles (2.5 cycles) has been shown between individual analysis and pool test with 8 samples (1/7 positive), respectively (Gupta et al, 2020). Similarly, another study showed that a positive sample of SARS-CoV-2 can be detected using TaqMan assays in pooled samples without compromising the assay sensitivity (Ben-Ami et al, 2020).…”

Section: Pooling Testmentioning

confidence: 98%

A comparative evaluation of a dye-based and probe-based RT-qPCR assay for the screening of SARS-CoV-2 using individual and pooled-sample testing

Verdugo

Plaza

Acosta‐Jamett

et al. 2020

Preprint

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Effective interventions are mandatory to control the transmission and spread of SARS-CoV-2, a highly contagious virus causing devastating effects worldwide. Cost-effective approaches are pivotal tools required to increase the detection rates and escalate further in massive surveillance programs, especially in countries with limited resources that most of the efforts have focused on symptomatic cases only. Here, we compared the performance of the RT-qPCR using an intercalating dye with the probe-based assay. Then, we tested and compared these two RT-qPCR chemistries in different pooling systems: after RNA extraction (post-RNA extraction) and before RNA extraction (pre-RNA extraction) optimizing by pool size and template volume. We evaluated these approaches in 610 clinical samples. Our results show that the dye-based technique has a high analytical sensitivity similar to the probe-based detection assay used worldwide. Further, this assay may also be applicable in testing by pool systems post-RNA extraction up to 20 samples. However, the most efficient system for massive surveillance, the pre-RNA extraction pooling approach, was obtained with the probe-based assay in test up to 10 samples adding 13.5 uL of RNA template. The low cost and the potential use in pre-RNA extraction pool systems, place of this assays as a valuable resource for scalable sampling to larger populations. Implementing a pool system for population sampling results in an important savings of laboratory resources and time, which are two key factors during an epidemic outbreak. Using the pooling approaches evaluated here, we are confident that it can be used as a valid alternative assay for the detection of SARS-CoV-2 in human samples.

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Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: a reliable, faster and economical method

Cited by 9 publications

References 11 publications

Effectiveness of Sample Pooling Strategies for SARS-CoV-2 Mass Screening by RT-PCR: A Scoping Review

Effectiveness of Sample Pooling Strategies for SARS-CoV-2 Mass Screening by RT-PCR: A Scoping Review

Pooling of coronavirus tests under unknown prevalence

A comparative evaluation of a dye-based and probe-based RT-qPCR assay for the screening of SARS-CoV-2 using individual and pooled-sample testing

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