RNase in the saliva can affect the detection of severe acute respiratory syndrome coronavirus 2 by real-time one-step polymerase chain reaction using saliva samples

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

Download Full-text

Low frequency of CYP2A6 gene polymorphism as revealed by a one-step polymerase chain reaction method

Pharmacogenetics ◽

10.1097/00008571-199906000-00007 ◽

1999 ◽

Vol 9 (3) ◽

pp. 327-332 ◽

Cited By ~ 37

Author(s):

Gen-Fu Chen ◽

Yong-Ming Tang ◽

Bridgett Green ◽

Dong-Xin Lin ◽

F. Peter Guengerich ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Gene Polymorphism ◽

Low Frequency ◽

Polymerase Chain Reaction Method ◽

Chain Reaction ◽

Reaction Method ◽

One Step ◽

Polymerase Chain ◽

Cyp2a6 Gene ◽

Step Polymerase Chain Reaction

Download Full-text

Multiplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assays for Diagnostic Testing of Severe Acute Respiratory Syndrome Coronavirus 2 and Seasonal Influenza Viruses: A Challenge of the Phase 3 Pandemic Setting

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa658 ◽

2020 ◽

Author(s):

Fabiola Mancini ◽

Fabrizio Barbanti ◽

Maria Scaturro ◽

Stefano Fontana ◽

Angela Di Martino ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Severe Acute Respiratory Syndrome ◽

Real Time ◽

Reverse Transcription ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Activity Period ◽

Clinical Samples ◽

Chain Reaction ◽

Polymerase Chain

Abstract Background Pandemic coronavirus disease 2019 (COVID-19) disease represents a challenge for healthcare structures. The molecular confirmation of samples from infected individuals is crucial and therefore guides public health decision making. Clusters and possibly increased diffuse transmission could occur in the context of the next influenza season. For this reason, a diagnostic test able to discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza viruses is urgently needed. Methods A multiplex real-time reverse-transcription polymerase chain reaction (PCR) assay was assessed using 1 laboratory protocol with different real-time PCR instruments. Overall, 1000 clinical samples (600 from samples SARS-CoV-2–infected patients, 200 samples from influenza-infected patients, and 200 negative samples) were analyzed. Results The assay developed was able to detect and discriminate each virus target and to intercept coinfections. The limit of quantification of each assay ranged between 5 and 10 genomic copy numbers, with a cutoff value of 37.7 and 37.8 for influenza and SARS-CoV-2 viruses, respectively. Only 2 influenza coinfections were detected in COVID-19 samples. Conclusions This study suggests that multiplex assay is a rapid, valid, and accurate method for the detection of SARS-CoV-2 and influenza viruses in clinical samples. The test may be an important diagnostic tool for both diagnostic and surveillance purposes during the seasonal influenza activity period.

Download Full-text

Presymptomatic Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 Among Residents and Staff at a Skilled Nursing Facility: Results of Real-time Polymerase Chain Reaction and Serologic Testing

Clinical Infectious Diseases ◽

10.1093/cid/ciaa991 ◽

2020 ◽

Cited By ~ 8

Author(s):

Scott A Goldberg ◽

Jochen Lennerz ◽

Michael Klompas ◽

Eden Mark ◽

Virginia M Pierce ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Severe Acute Respiratory Syndrome ◽

Real Time ◽

Skilled Nursing Facility ◽

Chain Reaction ◽

Nursing Facility ◽

Skilled Nursing ◽

Routine Testing ◽

Serologic Testing ◽

Polymerase Chain

Abstract High rates of asymptomatic coronavirus disease 2019 infection suggest benefits to routine testing in congregate care settings. Screening was undertaken in a single nursing facility without a known case of coronavirus disease 2019, demonstrating an 85% prevalence among residents and 37% among staff. Serology was not helpful in identifying infections.

Download Full-text

One-step polymerase chain reaction-based typing of Helicobacter pylori vacA gene: association with gastric histopathology

Medical Microbiology and Immunology ◽

10.1007/s004300050115 ◽

1999 ◽

Vol 188 (3) ◽

pp. 131-138 ◽

Cited By ~ 5

Author(s):

Shan-Rui Han ◽

Thomas Schneider ◽

Michael Loos ◽

Sucharit Bhakdi ◽

Markus J. Maeurer

Keyword(s):

Polymerase Chain Reaction ◽

Helicobacter Pylori ◽

Chain Reaction ◽

Gene Association ◽

One Step ◽

Polymerase Chain ◽

Vaca Gene ◽

Step Polymerase Chain Reaction

Download Full-text

Analysis of the persistence time of the SARS-CoV-2 virus in the cadaver and the risk of passing infection to autopsy staff

Medico-Legal Journal ◽

10.1177/0025817220980601 ◽

2021 ◽

pp. 002581722098060

Author(s):

Isabella Aquila ◽

Pietrantonio Ricci ◽

Carlo Filippo Bonetta ◽

Matteo Antonio Sacco ◽

Federico Longhini ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Reverse Transcription ◽

Post Mortem ◽

Forensic Pathologist ◽

Chain Reaction ◽

Sample Group ◽

Persistence Time ◽

One Step ◽

Polymerase Chain

The activity of the SARS-CoV-2 virus has not yet been studied in a post-mortem setting. The absence of these data has led to the prohibition of exposure of infected corpses during burial procedures. Our aim was to assess the virus’s persistence and the possibility of transmission in the post-mortem phase including autopsy staff. The sample group included 29 patients who were admitted to our Covid-19 Centre who died during hospitalisation and the autopsy staff. All the swabs were subjected to a one-step real-time reverse transcription polymerase chain reaction with cycle threshold (Ct) values. Swab collection was performed at 2 h, 4 h, 6 h, 12 h, over 24 since death. The following were the analysis of patients’ swabs: 10 cases were positive 2 h after death; 10 cases positive 4 h after death; 9 cases were found positive 6 h after death; 7 cases positive 12 h after death; 9 cases remained positive 24 h after death. The swabs performed on all the forensic pathologist staff on duty who performed the autopsies were negative. The choice to avoid rituals and the display of corpses before and at the burial procedures given appears cautiously valid due to the persistence of the SARS-CoV-2 virus in the post-mortem period. Although the caution in choosing whether or not to perform an autopsy on infected corpses is acceptable, not to perform autopsies is not biologically supported.

Download Full-text