Detection of pseudorabies virus with a real‐time recombinase‐aided amplification assay

Tu, Fei; Zhang, Yongning; Xu, Shengkui; Yang, Xintan; Zhou, Lei; Ge, Xinna; Han, Jun; Guo, Xin; Yang, Hanchun

doi:10.1111/tbed.14241

Cited by 16 publications

(20 citation statements)

References 26 publications

(39 reference statements)

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“…During latent infection of PRV, no infectious virions are produced, but the genome of the virus can be detected in the host. However, latent infection of the virus can be detected by serology, in situ hybridization, tissue co-culture, polymerase chain reaction (PCR), fluorescence quantitative PCR, and real-time recombinant enzyme-assisted amplification [ 46 , 109 , 110 , 111 , 112 , 113 ]. Similar to the PRV virulent strain, the PRV gene deletion vaccine can also establish latent infection in the PNS of pigs.…”

Section: Discussionmentioning

confidence: 99%

The Role of Latency-Associated Transcripts in the Latent Infection of Pseudorabies Virus

Deng

Liu

et al. 2022

Viruses

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Pseudorabies virus (PRV) can cause neurological, respiratory, and reproductive diseases in pigs and establish lifelong latent infection in the peripheral nervous system (PNS). Latent infection is a typical feature of PRV, which brings great difficulties to the prevention, control, and eradication of pseudorabies. The integral mechanism of latent infection is still unclear. Latency-associated transcripts (LAT) gene is the only transcriptional region during latent infection of PRV which plays the key role in regulating viral latent infection and inhibiting apoptosis. Here, we review the characteristics of PRV latent infection and the transcriptional characteristics of the LAT gene. We also analyzed the function of non-coding RNA (ncRNA) produced by the LAT gene and its importance in latent infection. Furthermore, we provided possible strategies to solve the problem of latent infection of virulent PRV strains in the host. In short, the detailed mechanism of PRV latent infection needs to be further studied and elucidated.

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Section: Discussionmentioning

confidence: 99%

The Role of Latency-Associated Transcripts in the Latent Infection of Pseudorabies Virus

Deng

Liu

et al. 2022

Viruses

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“…Here, we developed a novel real-time RT-RAA assay targeting the N gene (highly conserved) for the rapid detection of SARS-CoV-2. We adopted the primer screening strategy reported in previous studies [ 11 ]. Briefly, we used a forward primer (randomly selected) to screen all of the reverse primers, and then the best reverse primer was selected and used to screen all of the forward primers.…”

Section: Discussionmentioning

confidence: 99%

“…Recombinase-aided amplification (RAA), a new isothermal amplification technique, can be completed within 30 min at 37–42 °C. There are three key enzymes in the RAA system: a single-stranded DNA-binding protein (SSB), which is a protein specifically responsible for binding to single-stranded regions of DNA; a recombinase, which pairs the specific primers with template DNA; and a strand-displacing DNA polymerase for extension and DNA amplification [ 10 , 11 ]. In the RAA system, the addition of specific fluorescent probes can be used for real-time monitoring of DNA amplicons.…”

Section: Introductionmentioning

confidence: 99%

Real-Time Reverse Transcription Recombinase-Aided Amplification Assay for Rapid Amplification of the N Gene of SARS-CoV-2

Tu¹,

Zhang

Zhang³

et al. 2022

IJMS

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COVID-19 was officially declared a global pandemic disease on 11 March 2020, with severe implications for healthcare systems, economic activity, and human life worldwide. Fast and sensitive amplification of the severe acute respiratory syndrome virus 2 (SARS-CoV-2) nucleic acids is critical for controlling the spread of this disease. Here, a real-time reverse transcription recombinase-aided amplification (RT-RAA) assay, targeting conserved positions in the nucleocapsid protein gene (N gene) of SARS-CoV-2, was successfully established for SARS-CoV-2. The assay was specific to SARS-CoV-2, and there was no cross-reaction with other important viruses. The sensitivity of the real-time RT-RAA assay was 142 copies per reaction at 95% probability. Furthermore, 100% concordance between the real-time RT-RAA and RT-qPCR assays was achieved after testing 72 clinical specimens. Further linear regression analysis indicated a significant correlation between the real-time RT-RAA and RT-qPCR assays with an R2 value of 0.8149 (p < 0.0001). In addition, the amplicons of the real-time RT-RAA assay could be directly visualized by a portable blue light instrument, making it suitable for the rapid amplification of SARS-CoV-2 in resource-limited settings. Therefore, the real-time RT-RAA method allows the specific, sensitive, simple, rapid, and reliable detection of SARS-CoV-2.

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“…In recent years, it has been used for the agricultural detection of pathogens, and the method has shown specific and sensitive performance [ 37 , 38 ]. Tu reported on the application of swine fever virus and porcine pseudorabies virus in RT-RAA [ 39 , 40 ]. Wang reported on the detection of Newcastle disease virus and infectious bronchitis virus in chickens by RT-RAA [ 41 , 42 ].…”

Section: Introductionmentioning

confidence: 99%

Development and Application of a Reverse-Transcription Recombinase-Aided Amplification Assay for Porcine Epidemic Diarrhea Virus

Liu

Gao

et al. 2022

Viruses

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Porcine epidemic diarrhea virus (PEDV) is a coronavirus currently widespread worldwide in the swine industry. Since PEDV was discovered in China in 1984, it has caused huge economic losses in the swine industry. PEDV can infect pigs of all ages, but piglets have the highest infection with a death rate as high as 100%, and the clinical symptoms are watery diarrhea, vomiting, and dehydration. At present, there is not any report on PEDV detection by RT-RAA. In this study, we developed an isothermal amplification technology by using reverse-transcription recombinase-aided amplification assay (RT-RAA) combined with portable instruments to achieve a molecular diagnosis of PEDV in clinical samples from China. By designing a pair of RT-RAA primers and probes based on the PEDV N gene, this method breaks the limitations of existing detection methods. The assay time was within 30 min at 41 °C and can detect as few as 10 copies of PEDV DNA molecules per reaction. Sixty-two clinical tissue samples were detected by RT-qPCR and RT-RAA. The positive and negative rates for the two methods were 24.19% and 75.81%, respectively. Specificity assay showed that the RT-RAA had specifically detected PEDV and was not reactive for porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), swine flu virus (SIV), or porcine Japanese encephalitis virus (JEV). The results suggested that RT-RAA had a strong specificity and high detection sensitivity when combined with a portable instrument to complete the detection under a constant temperature of 30 min, which are more suitable for preventing and controlling PEDV onsite in China.

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Detection of pseudorabies virus with a real‐time recombinase‐aided amplification assay

Cited by 16 publications

References 26 publications

The Role of Latency-Associated Transcripts in the Latent Infection of Pseudorabies Virus

The Role of Latency-Associated Transcripts in the Latent Infection of Pseudorabies Virus

Real-Time Reverse Transcription Recombinase-Aided Amplification Assay for Rapid Amplification of the N Gene of SARS-CoV-2

Development and Application of a Reverse-Transcription Recombinase-Aided Amplification Assay for Porcine Epidemic Diarrhea Virus

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