scholarly journals Viroselect: A novel SARS-CoV-2 detection assay to resolve inconclusive samples

2021 ◽  
Author(s):  
Ketki Jawade ◽  
Akhauri Yash Sinha ◽  
Sharad Bhagat ◽  
Shilpa Bhowmick ◽  
Bhagyashree Chauhan ◽  
...  
Keyword(s):  
Retrospective Analysis ◽  
High Throughput ◽  
Hypervariable Region ◽  
Structural Protein ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Target Region ◽  
Detection Assay ◽  
High Concordance ◽  
Reporting Delays

ABSTRACTBackgroundIndia bears the second largest burden of SARS-CoV-2 infection. A multitude of RT-PCR detection assays with disparate gene targets including automated high throughput platforms are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays leading to suboptimal disease management. Here, we report the development of a novel ORF-1a based SARS-CoV-2 RT-PCR assay, Viroselect, showing high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India.MethodsWe identified a unique target region within SARS-CoV-2 ORF1a, non-structural protein (nsp3), that was used to design and develop our assay. This hypervariable region (1933-3956) between SARS-CoV-2, SARS-CoV, and MERS-COV was utilized to design our primers and probe for RT-PCR assay. We further evaluated concordance of our assay with commonly used EUA (USFDA) manual kits as well as an automated high throughput testing platform. Further, a retrospective analysis using Viroselect on samples reported as ‘inconclusive’ during April-October 2020 was carried out.ResultsA total of 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect assay with both manual (87.6%; 95% CI) as well as automated (84.7%; 95% CI) testing assays. Also, in the retrospective analysis of 224 additional samples reported as ‘inconclusive’, Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) samples which were termed inconclusive by manual and automated high throughput platform respectively.ConclusionWe show that Viroselect had high concordance with conventional assays, both manual and automated, as well as highlight its potential in resolving inconclusive samples.

scholarly journals High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247115
Author(s):  
Rahul C. Bhoyar ◽  
Abhinav Jain ◽  
Paras Sehgal ◽  
Mohit Kumar Divakar ◽  
Disha Sharma ◽  
...  
Keyword(s):  
High Throughput ◽  
Genetic Epidemiology ◽  
Confirmatory Test ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Additional Advantage ◽  
Depth Analysis ◽  
High Concordance ◽  
High Throughput Detection ◽  
First Time

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.

Development of a high-throughput multiplexed real time RT-PCR assay for detection of human pegivirus 1 and 2

2017 ◽  
Vol 241 ◽  
pp. 34-40 ◽  
Author(s):  
Matthew Frankel ◽  
Kenn Forberg ◽  
Kelly E. Coller ◽  
Michael G. Berg ◽  
John Hackett ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Rt Pcr ◽  
Pcr Assay

scholarly journals Development and evaluation of a SYBR Green I RT-qPCR assay for Feline Infection Peritonitis virus detection

2020 ◽  
Author(s):  
Yuanhong Wang ◽  
Yong Wang ◽  
Jing Wang ◽  
Liang Zhao ◽  
Chuanfeng Li ◽  
...  
Keyword(s):  
Systemic Disease ◽  
Virus Detection ◽  
Sybr Green ◽  
Sybr Green I ◽  
Mutant Form ◽  
Structural Protein ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Feline Infectious Peritonitis ◽  
Viral Loads

Abstract Background Feline Infectious Peritonitis (FIP) is a fatal, systemic disease caused by a mutant form of Feline Infectious Peritonitis virus (FIPV) and has been reported to occur worldwide in domestic cats and massive wild feline species. Meanwhile a definitive diagnosis of FIPV ante mortem remains challenging. The objective was to develop a qPCR for the detection of FIPV in cats and applied the assay to detected the viral loads in different autopsied organs of a cat naturally infected with FIPV. Results: After genetic comparison, We develop a SYBR Green I based quantitative transcription PCR assay (qPCR) targeting the structural protein N of FIPV. The sensitivity of the new assay in detecting FIPV nucleic acids was approximately 1000 times higher than that of the conventional RT-PCR assay (PCR). There were no cross-reactions with other common viruses. Organ assay showed that FIPV were present in the Heart, liver, spleen, lung, kidney, duodenal and ascites of the autopsied cat. Histological lesions showed that macrophages, non-toxic neutrophils and lymphocytes predominated in different organs which confirmed that the cat was infected with FIPV. Conclusions We developed a quantitative platform for epidemiological investigations study of FIPV that was simple, sensitive, and rapid.

scholarly journals Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

2011 ◽  
Vol 78 (1) ◽  
pp. 156-162 ◽  
Author(s):  
Anne M. Johnson ◽  
George D. Di Giovanni ◽  
Paul A. Rochelle
Keyword(s):  
Cell Culture ◽  
Drinking Water ◽  
Cryptosporidium Parvum ◽  
False Positives ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Cryptosporidium Hominis ◽  
Content Type ◽  
Detection Assay ◽  
Pcr Targeting

ABSTRACTThis study compared the three most commonly used assays for detectingCryptosporidiumsp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targetingCryptosporidiumsp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targetingCryptosporidiumsp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumeratedCryptosporidium parvumoocysts, including infection with one oocyst and three oocysts. All methods also detected infection withCryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with threeC. parvumoocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectiousCryptosporidium parvumandCryptosporidium hominisin drinking water.

scholarly journals Novel automated sample-to-result SARS-CoV-2 laboratory-developed RT-PCR assay for high-throughput testing using LabTurbo AIO 48 system

2021 ◽  
Vol 514 ◽  
pp. 54-58
Author(s):  
Ming-Jr Jian ◽  
Hsing-Yi Chung ◽  
Chih-Kai Chang ◽  
Jung-Chung Lin ◽  
Kuo-Ming Yeh ◽  
...  
Keyword(s):  
High Throughput ◽  
Rt Pcr ◽  
Pcr Assay

scholarly journals Validation of a SARS‐CoV‐2 RNA RT‐PCR assay for high‐throughput testing in blood of COVID ‐19 convalescent plasma donors and patients

Transfusion ◽  
2020 ◽  
Author(s):  
Erwin F. Strasser ◽  
Philipp A. Steininger ◽  
Klaus Korn ◽  
Susanne Achenbach ◽  
Matthias Tenbusch ◽  
...  
Keyword(s):  
High Throughput ◽  
Rt Pcr ◽  
Pcr Assay

scholarly journals High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

10.3791/52650 ◽  
2015 ◽  
Author(s):  
Lynnsey A. Renn ◽  
Terence C. Theisen ◽  
Maria B. Navarro ◽  
Viraj P. Mane ◽  
Lynnsie M. Schramm ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Expression Profiles ◽  
Rt Pcr ◽  
Pcr Assay
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