Development of a high-throughput RT-PCR based viral infectivity assay for monitoring the stability of a replicating recombinant Lymphocytic Choriomeningitis viral vector

We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. The high sensitivity and large dynamic range of our cost effective assay alleviates the need for serial dilution experiments.

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Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

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Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction

BioTechniques ◽

10.2144/05382rr05 ◽

2005 ◽

Vol 38 (2) ◽

pp. 287-293 ◽

Cited By ~ 183

Author(s):

Van Luu-The ◽

Nathalie Paquet ◽

Ezequiel Calvo ◽

Jean Cumps

Keyword(s):

Real Time ◽

High Throughput ◽

Second Derivative ◽

Rt Pcr ◽

Pcr Method

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Effect of pH and temperature on the infectivity of human coronavirus 229E

Canadian Journal of Microbiology ◽

10.1139/m89-160 ◽

1989 ◽

Vol 35 (10) ◽

pp. 972-974 ◽

Cited By ~ 40

Author(s):

Alain Lamarre ◽

Pierre J. Talbot

Keyword(s):

Incubation Period ◽

Storage Conditions ◽

Viral Infectivity ◽

Human Coronavirus ◽

Virus Growth ◽

The Stability ◽

And Storage ◽

Influence Of Ph ◽

Human Coronavirus 229E

The stability of human coronavirus 229E infectivity was maximum at pH 6.0 when incubated at either 4 or 33 °C. However, the influence of pH was more pronounced at 33 °C. Viral infectivity was completely lost after a 14-day incubation period at 22, 33, or 37 °C but remained relatively constant at 4 °C for the same length of time. Finally, the infectious titer did not show any significant reduction when subjected to 25 cycles of thawing and freezing. These studies will contribute to optimize virus growth and storage conditions, which will facilitate the molecular characterization of this important pathogen.Key words: coronavirus, pH, temperature, infectivity, human coronavirus.

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Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽

10.1094/pdis-05-21-0949-re ◽

2021 ◽

Author(s):

Dan Edward Veloso Villamor ◽

Karen E Keller ◽

Robert Martin ◽

Ioannis Emmanouil Tzanetakis

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Wide Spectrum ◽

Virus Detection ◽

Rt Pcr ◽

Host Interactions ◽

Growing Seasons ◽

Berry Crops ◽

Sampling Times ◽

Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

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Multiplex Detection of Rotavirus A, B, C, Norovirus GI and GII, Adenovirus, Astrovirus and Sapovirus in a Single PCR System in Stool Samples From Acute Diarrhea Children

10.21203/rs.3.rs-111216/v1 ◽

2020 ◽

Author(s):

Wei Li ◽

Weiwei Li ◽

Ran Tao ◽

Wenqing Xiang ◽

Mingming Zhou ◽

...

Keyword(s):

High Throughput ◽

Acute Diarrhea ◽

Cross Reaction ◽

Melting Curve Analysis ◽

Clinical Test ◽

Rt Pcr ◽

Rotavirus A ◽

Diarrhea Virus ◽

Intestinal Pathogens ◽

Stool Samples

Abstract Background: Early and accurate identification of infection viruses among children can benefit the personalized medical treatment and management, and reduce the future occurrence of serious symptoms. Thus, it is critical to develop a high-throughput multiplex real-time RT-PCR method to improve the accuracy and efficiency in routine clinical lab tests. Here, we developed a RT-PCR combined with melting curve analysis (RRCMC) method for simultaneous detection of rotavirus A, B, C, norovirus GI and GII, adenovirus, astrovirus and sapovirus. Results: Stool samples were collected from 160 children with acute diarrhea and tested by RRCMC assay. A total of 71 patients were tested positive with norovirus, adenovirus or rotavirus. The RRCMC assay has high specificity. There is no internal cross-reaction through the 8 diarrhea viruses and no cross-reaction of other commonly intestinal pathogens and human genome. The detection limit was ranging from 1×102 to 1×105 copies/ml for each diarrhea virus. Conclusions: In conclusion, the RRCMC method is a suitable rapid clinical test for infection viruses, with the advantages of high-throughput, low cost, high sensitivity, and specificity.

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Expression profile of microRNAs in gastrointestinal stromal tumors revealed by high throughput quantitative RT-PCR microarray

World Journal of Gastroenterology ◽

10.3748/wjg.v21.i19.5843 ◽

2015 ◽

Vol 21 (19) ◽

pp. 5843-5855 ◽

Cited By ~ 13

Author(s):

Han-Xing Tong ◽

Yu-Hong Zhou ◽

Ying-Yong Hou ◽

Yong Zhang ◽

Yuan Huang ◽

...

Keyword(s):

Expression Profile ◽

High Throughput ◽

Gastrointestinal Stromal Tumors ◽

Rt Pcr ◽

Stromal Tumors ◽

Pcr Microarray

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High-throughput immunoassays for SARS-CoV-2, considerable differences in performance when comparing three methods

10.1101/2020.05.22.20106294 ◽

2020 ◽

Cited By ~ 1

Author(s):

Oskar Ekelund ◽

Kim Ekblom ◽

Sofia Somajo ◽

Johanna Pattison-Granberg ◽

Karl Olsson ◽

...

Keyword(s):

High Throughput ◽

Rapid Test ◽

Performance Criteria ◽

Clinical Use ◽

Rt Pcr ◽

Regulatory Authorities ◽

Flow Test ◽

Lateral Flow Test ◽

High Level ◽

Positive Results

Background: The recently launched high-throughput assays for the detection of antibodies against SARS-CoV-2 may change the managing strategies for the COVID-19 pandemic. This study aimed at investigating the performance of three high-throughput assays and one rapid lateral flow test relative to the recommended criteria defined by regulatory authorities. Methods: A total of 133 samples, including 100 pre-pandemic samples, 20 samples from SARS-CoV-2 RT-PCR positive individuals, and 13 potentially cross-reactive samples were analysed with SARS-CoV-2 IgG (Abbott), Elecsys Anti-SARS-CoV-2 (Roche), LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin) and 2019-nCOV IgG/IgM Rapid Test (Dynamiker Biotechnology Co). Results: All assays performed with a high level of specificity; however, only Abbott reached 100% (95% CI 96.3-100). The pre-pandemic samples analysed with Roche, DiaSorin and Dynamiker Biotechnology resulted in two to three false-positive results per method (specificity 96.9-98.0%). Sensitivity differed more between the assays, Roche exhibiting the highest sensitivity (100%, CI 83.9-100). The corresponding figures for Abbott, DiaSorin and Dynamiker Biotechnology were 85.0%, 77.8% and 75.0%, respectively. Conclusions: The results of the evaluated SARS-CoV-2 assays vary considerably as well as their ability to fulfil the performance criteria proposed by regulatory authorities. Introduction into clinical use in low-prevalent settings, should therefore, be made with caution.

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Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽

10.1371/journal.pone.0252687 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252687

Author(s):

Sukalyani Banik ◽

Kaheerman Saibire ◽

Shraddha Suryavanshi ◽

Glenn Johns ◽

Soumitesh Chakravorty ◽

...

Keyword(s):

Limit Of Detection ◽

Viral Rna ◽

Clinical Samples ◽

Sample Collection ◽

Rt Pcr ◽

Diagnostic Assays ◽

Guanidinium Thiocyanate ◽

Upper Respiratory ◽

Polymerase Chain ◽

The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

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High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v1 (protocols.io.btywnpxe)

protocols.io ◽

10.17504/protocols.io.btywnpxe ◽

2021 ◽

Author(s):

Aaron Topol ◽

marlene.wolfe not provided ◽

Brad White ◽

Krista Wigginton ◽

Alexandria B

Keyword(s):

High Throughput ◽

Rt Pcr

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