SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses

Escutenaire, Sophie; Mohamed, Nik Abdullah Nik; Isaksson, Mats; Thorén, Peter; Klingeborn, B.; Belák, Sándór; Berg, Mikael; Blomberg, Jonas

doi:10.1007/s00705-006-0840-x

Cited by 87 publications

(114 citation statements)

References 37 publications

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“…Even now, the viral titer of PEDV in cell culture is still low. Other diagnostic methods for detecting PEDV include immunohistochemistry, in situ hybridization, dot-blot hybridization, RT-PCR, and real-time RT-PCR [24][25][26][27][28]. These methods may require either high-precision instruments or complicated procedures.…”

Section: Discussionmentioning

confidence: 99%

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus

Ren

2011

Virus Genes

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In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detection of porcine epidemic diarrhea virus (PEDV). Six primers were designed to amplify the nucleocapsid (N) gene of PEDV. The optimization, sensitivity, and specificity of the RT-LAMP were investigated. The results showed that the optimal reaction condition for RT-LAMP amplifying PEDV N gene was achieved at 63°C for 50 min. The RT-LAMP assay was more sensitive than gel-based RT-PCR and enzyme-linked immunosorbent assay. It was capable of detecting PEDV from clinical samples and differentiating PEDV from Porcine transmissible gastroenteritis virus, Porcine rotavirus, Porcine pseudorabies virus, Porcine reproductive and respiratory syndrome virus, and Avian infectious bronchitis virus.

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Section: Discussionmentioning

confidence: 99%

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus

Ren

2011

Virus Genes

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“…132 When a coronavirus etiology is suspected, pan-coronavirus detection methods are very useful to the diagnostician. One such tool is the consensus coronavirus RT-PCR assay, developed by Escutenaire et al 32 This RT-PCR assay targets a 179-bp region of the open reading frame (ORF) 1b of the replicase gene. The assay, designed for real-time detection using SYBR Green chemistry, was validated with a mix of 36 human or animal coronavirus strains, covering the alpha-, beta-, and gammacoronavirus genera.…”

Section: Coronavirusesmentioning

confidence: 99%

Beyond H&E

et al. 2013

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Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets. Keywords cancer, ISH, IHC, nucleic acid analysis, molecular pathology, PCR, virology Technical ConsiderationsTissue Handling, Fixation, and Embedding Fixation of tissues in 10% neutral buffered formalin, followed by paraffin embedding (FFPE), has long been the standard approach of tissue preparation for microscopic evaluation. The chemical principle of formalin fixation is based on the crosslinking of proteins. Formalin has been used extensively because it is inexpensive and excellently preserves morphological detail in histological sections. However, the relatively recent development and increasing use of both protein and nucleic acid detection methods have revealed some significant shortcomings associated with the formalin-based fixation process. The effects of formalin fixation on DNA are chemical modification, DNA trapping, and potentially DNA fragmentation. Chemical modification, considered the main effect, results from the direct cross-linking of proteins to DNA. 68 In addition, extensive protein-protein cross-linking and the formation of protein networks result in DNA trapping. The combination of these effects makes it much more difficult to extract DNA from FFPE tissue than from unfixed tissue, especially when methods used for extraction from fresh tissues are used on FFPE tissue without any modifications. 60,83 In addition to interfering with the ability to extract nucleic acid, formalin fixation also causes DNA fragmentation, especially when the formalin used is inadequately buffered, which then leads to formic acid formation during the fixation process. This, in turn, results in depurination of the DNA, which then becomes susceptible to cleavage by hydroxyl ions and results in the formation of single-stranded breaks.60 Similar to DNA, RNA is also degraded and chemically modified as the result of formalin fixation. The most important chemical effect of formalin on RNA is the formation of monomethylol groups with purine bases. 77 This has a significant effect on reverse transcription ...

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“…Standard serological tests that are used to detect TGEV also require considerable time to complete, and have not been able to distinguish between different strains. The results can be obtained using RT-PCR more rapidly than with the VI method or serological tests, and the RT-PCR technique has been used increasingly as a supplementary method in TGEV diagnosis [19][20][21][22][23][24][25][26][27]. Paton et al [25] detected TGEV in clinical fecal specimens using single-round PCR and was able to discriminate TGEV from PRCV.…”

Section: Introductionmentioning

confidence: 99%

Rapid differentiation of vaccine strain and Chinese field strains of transmissible gastroenteritis virus by restriction fragment length polymorphism of the N gene

et al. 2010

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A strain of transmissible gastroenteritis virus (TGEV), designated H16, was isolated in PK-15 cells and passaged serially to level 165. Vaccines based on passages 155-165 in cell cultures are available commercially as vaccines for the prevention and control of infections with TGEV in China. Nucleoprotein (N) sequences of the virus at passages 155 and 165 were aligned and compared using a computer software program. The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiation of the vaccine strain from the other TGEVs was investigated. The RFLP analysis identified a change in the cleavage sites of AclI at passages 155 and 165. This RFLP pattern of the N gene differentiated the Chinese vaccine strain from its parental strain, the 11 TGEVs studied and the other reported TGEVs in the GenBank. Using phylogenetic analysis, the Chinese TGEVs were divided into three groups (G1, G2, and G3). The G3 Chinese TGEVs possessed several specific nucleotides and amino acids that were not found in the G1 and G2 Chinese TGEVs or the other reference TGEVs. Analysis of the phylogenetic trees revealed that the G3 TGEVs represent a separate group that is distinct from the non-Chinese TGEVs and from Chinese TGEVs isolated previously. These findings suggest that Chinese strains of TGEV are evolving continuously.

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SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses

Cited by 87 publications

References 37 publications

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus

Beyond H&E

Rapid differentiation of vaccine strain and Chinese field strains of transmissible gastroenteritis virus by restriction fragment length polymorphism of the N gene

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