A RT-PCR assay for the detection of coronaviruses from four genera

Xiu, Leshan; Binder, Raquel A.; Alarja, Natalie A; Kochek, Kara; Coleman, Kristen K.; Than, Son T.; Bailey, Emily S.; Bui, Vuong Nghia; Toh, Teck Hock; Erdman, Dean D.; Gray, Gregory C.

doi:10.1016/j.jcv.2020.104391

Cited by 45 publications

(66 citation statements)

References 19 publications

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“…Recently, there was a publication that introduced an RT‐PCR assay for the detection of coronaviruses from four genera with the LOD ranged from 4 to 4 × 10 2 copies/reaction, depending on the CoV species tested. This assay detected all previously known CoVs, including SARS‐CoV‐2, but it could not distinguish SARS‐CoV with SARS‐CoV‐2 20 …”

Section: Discussionmentioning

confidence: 92%

A simple method for detection of a novel coronavirus (SARS‐CoV‐2) using one‐step RT‐PCR followed by restriction fragment length polymorphism

Sơn

Hang

Thuan

et al. 2020

Journal of Medical Virology

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A novel coronavirus associated with acute respiratory disease (named SARS-CoV-2) is recently identified in Wuhan city, China, spread rapidly worldwide. Early identification of this novel coronavirus by molecular tools is critical for surveillance and control of the epidemic outbreak. We aimed to establish a simple method for the detection of SARS-CoV-2 in differentiating with SARS-CoV. Primers of our in-house reverse transcription polymerase chain reaction (RT-PCR) assays were designed to target conserved regions of the RdRP gene and E gene, selected restriction enzymes EcoRI, Tsp45I, and AluI to distinguish between SARS-CoV-2 and SARS-CoV. In this report, a 396-bp fragment of the RdRp gene and 345-bp fragment of the E gene were amplified by one-step RT-PCR. Enzyme Tsp45I cuts the RdRP-amplified product of SARS-CoV-2 generating three fragments of 45, 154, and 197 bp, but it did not cut the amplicon of SARS-CoV. In contrast, the amplified product of SARS-CoV was digested with EcoRI producing two fragments of 76 and 320 bp, whereas the amplicon of SARS-CoV-2 was undigested by Tsp45I help to distinguish clearly SARS-CoV-2 from SARS-CoV on gel electrophoresis. In addition, AluI cut the amplicon of the E gene of SARS-CoV-2 generating two fragments of 248 and 97 bp without cutting to SARS-CoV. The accuracy of the assay was confirmed by sequencing and phylogenetic analysis. When evaluated on clinical samples showed a high sensitivity of 95%, specificity of our assay was 100% and clinical performance for detection of SARS-CoV-2 in comparison with other reference assays. In conclusion, in the present study, we successfully developed a simple method for molecular detection of SARS-CoV-2 in differentiating with SARS-CoV.

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Section: Discussionmentioning

confidence: 92%

A simple method for detection of a novel coronavirus (SARS‐CoV‐2) using one‐step RT‐PCR followed by restriction fragment length polymorphism

Sơn

Hang

Thuan

et al. 2020

Journal of Medical Virology

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“…For now after all efforts diagnosis for SARS-CoV-2 has been found but as lots of virus under this family can emerge in future and which has already spread in past needed to be studied [76]. So one research was completely done to make a molecular diagnostic kit which can identify almost all CoVs, thus to diagnose easily any virus among this family in future [77]. They designed a semi-nested RT-PCR based upon 38 genome sequences that has been recorded from human and animal CoVs.…”

Section: Future Prospects Of Rt-pcrmentioning

confidence: 99%

Development of RT-PCR Based Diagnosis of SARS-CoV-2

Patankar¹,

Zambare²

2022

Biotechnology to Combat COVID-19

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In the 2020, COVID-19 pandemic disease created an havoc situation world widely and mainly caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). It has been challenging task for researchers, scientists and medico-pharmaceutical organisations to find out rapid and reliable diagnosis methods. Among the all testing services, a Reverse Transcription Polymerase Chain Reaction (RT-PCR) is the more accurate, rapid and authenticated molecular technique used for most of the diagnosis of major diseases. It has been a global priority to fix the rapid diagnosis method to combat against the pandemic COVID-19. Thus, the present chapter mainly focussing on the progress of RT-PCR method development though various processes of data collection on isolation of whole genome sequence, its primer and method designing. In this scenario, India suddenly become the global leader for vaccine development and hence the challenges and RT-PCR kit development in India and rest of the world has been be discussed. World wide many Government and private agencies and industries have taken an initiative for diagnosis of SARS-CoV-2 hence this chapter also summarised the scope of RT-PCR to combat pandemic situation in future.

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“…Betacoronaviruses were thought to mainly infect animals and cause occasional mild infections in humans [ 1 , 3 ] until the outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), two highly infectious zoonotic diseases that caused major epidemics worldwide [ 3 , 4 ]. These viruses, along with other genetically diverse SARS-related coronaviruses (SARSr-CoV) and MERS-related coronaviruses (MERSr-CoV), have been transmitted from their natural hosts such as bats and rodents to an intermediate animal host of different species such as civet cats and dromedary camels before being transmitted to a human host [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. Genetically diverse coronaviruses have been known to cause infections not only in humans but also in animals [ 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

“…Genetically diverse coronaviruses have been known to cause infections not only in humans but also in animals [ 10 , 11 ]. Given the predisposition of coronaviruses, particularly betacoronaviruses, to switch hosts [ 5 ], an emergence or re-emergence of coronavirus disease or the possibility of another pandemic is highly plausible.…”

Section: Introductionmentioning

confidence: 99%

“…The emergence of the novel SARS-CoV-2 with its efficient human-to-human transmission and ability to cause asymptomatic infections [ 5 , 14 , 16 , 17 , 18 ] has highlighted the need to develop a highly specific and sensitive viral detection method to mitigate the potential contact with infected individuals and prevent the further spread of infection. At present, real-time quantitative polymerase chain reaction (RT-qPCR) is widely used to detect SARS-CoV-2 and plays a critical role in viral containment [ 13 , 19 , 20 , 21 ].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Peptide Nucleic Acid (PNA)-Enhanced Specificity of a Dual-Target Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Assay for the Detection and Differentiation of SARS-CoV-2 from Related Viruses

et al. 2020

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The threat posed by coronaviruses to human health has necessitated the development of a highly specific and sensitive viral detection method that could differentiate between the currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related coronaviruses (SARSr-CoVs). In this study, we developed a peptide nucleic acid (PNA)-based real-time quantitative polymerase chain reaction (RT-qPCR) assay targeting the N gene to efficiently discriminate SARS-CoV-2 from other SARSr-CoVs in human clinical samples. Without compromising the sensitivity, this method significantly enhanced the specificity of SARS-CoV-2 detection by 100-fold as compared to conventional RT-qPCR. In addition, we designed an RT-qPCR method for the sensitive and universal detection of ORF3ab-E genes of SARSr-CoV with a limit of detection (LOD) of 3.3 RNA copies per microliter. Thus, the developed assay serves as a confirmative dual-target detection method. Our PNA-mediated dual-target RT-qPCR assay can detect clinical SARS-CoV-2 samples in the range of 18.10–35.19 Ct values with an 82.6–100% detection rate. Furthermore, our assay showed no cross-reactions with other coronaviruses such as human coronaviruses (229E, NL63, and OC43) and Middle East respiratory syndrome coronavirus, influenza viruses (Type B, H1N1, H3N2, HPAI H5Nx, and H7N9), and other respiratory disease-causing viruses (MPV, RSV A, RSV B, PIV, AdV, and HRV). We, thus, developed a PNA-based RT-qPCR assay that differentiates emerging pathogens such as SARS-CoV-2 from closely related viruses such as SARSr-CoV and allows diagnosis of infections related to already identified or new coronavirus strains.

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A RT-PCR assay for the detection of coronaviruses from four genera

Cited by 45 publications

References 19 publications

A simple method for detection of a novel coronavirus (SARS‐CoV‐2) using one‐step RT‐PCR followed by restriction fragment length polymorphism

A simple method for detection of a novel coronavirus (SARS‐CoV‐2) using one‐step RT‐PCR followed by restriction fragment length polymorphism

Development of RT-PCR Based Diagnosis of SARS-CoV-2

Peptide Nucleic Acid (PNA)-Enhanced Specificity of a Dual-Target Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Assay for the Detection and Differentiation of SARS-CoV-2 from Related Viruses

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