The Development of a Real-Time Recombinase-Aid Amplification Assay for Rapid Detection of African Swine Fever Virus

Wu, Yongshu; Yang, Yang; Ru, Yi; Qin, Xiaodong; Li, Miaomiao; Zhang, Zhixiong; Zhang, Rui; Li, Yijing; Zhang, Zhidong; Li, Yanmin

doi:10.3389/fmicb.2022.846770

Cited by 5 publications

(3 citation statements)

References 28 publications

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“…Beyond that, the ASFV E248R gene was selected to be the target for establishing a real-time PCR method, which is used for the efficient detection of infected ASFV and PRRSV live vector viruses expressing ASFV antigen protein ( Li L. et al, 2022 ). Wu et al (2022) developed a real-time recombinase-aid amplification (RAA) assay to rapidly detect the different genotypes of ASFV, including the E70 strain (Spanish), the Anhui XCGQ strain, and the Georgia 2007/1 strain and contributed to the development of a control strategy for ASF ( Wu et al, 2022 ). These details could additionally shed light on potential protocols to prevent and control ASF.…”

Section: Discussionmentioning

confidence: 99%

Vaccines for African swine fever: an update

Zhang¹,

Zhao²,

Zhang³

et al. 2023

Front. Microbiol.

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African swine fever (ASF) is a fatal infectious disease of swine caused by the African swine fever virus (ASFV). Currently, the disease is listed as a legally notifiable disease that must be reported to the World Organization for Animal Health (WOAH). The economic losses to the global pig industry have been insurmountable since the outbreak of ASF. Control and eradication of ASF are very critical during the current pandemic. Vaccination is the optimal strategy to prevent and control the ASF epidemic, but since inactivated ASFV vaccines have poor immune protection and there aren’t enough cell lines for efficient in vitro ASFV replication, an ASF vaccine with high immunoprotective potential still remains to be explored. Knowledge of the course of disease evolution, the way of virus transmission, and the breakthrough point of vaccine design will facilitate the development of an ASF vaccine. In this review, the paper aims to highlight the recent advances and breakthroughs in the epidemic and transmission of ASF, virus mutation, and the development of vaccines in recent years, focusing on future directions and trends.

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Section: Discussionmentioning

confidence: 99%

Vaccines for African swine fever: an update

Zhang¹,

Zhao²,

Zhang³

et al. 2023

Front. Microbiol.

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“…Considering the convenience, sensitivity, and rapidity of identifying clinical viral infections in pigs, several technologies, such as PCR, real-time PCR, and digital PCR, are frequently used. There are also some articles about isothermal nucleic acid amplification, such as recombinase polymerase amplification (RPA), recombinase-aided amplification (RAA), and loop-mediated isothermal amplification (LAMP) [ 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. Combining CRISPR technology with flow immunochromatography to detect viruses has yielded positive results in some studies [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022

Liu

Zou

Liu

et al. 2023

Veterinary Sciences

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African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are important DNA viruses that cause reproductive disorders in sows, which result in huge losses in pig husbandry, especially in China. The multiplex qPCR assay could be utilized as a simultaneous diagnostic tool for field-based surveillance and the control of ASFV, PCV2, and PRV. Based on the conserved regions on the p72 gene of ASFV, the Cap gene of PCV2, the gE gene of PRV, and the porcine endogenous β-Actin gene, the appropriate primers and probes for a multiplex TaqMan real-time PCR test effective at concurrently detecting three DNA viruses were developed. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to swine reproductive diseases. In addition, its sensitivity was great, with a detection limit of 101 copies/L of each pathogen, and its repeatability was excellent, with intra- and inter-group variability coefficients of <2%. Applying this assay to detect 383 field specimens collected from 2020 to 2022, the survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV+ PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. Overall, the assay established in this study provides an effective tool for quickly distinguishing the viruses causing sow reproductive disorders, suggesting its huge clinical application value in the diagnosis of swine diseases.

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“…RAA has emerged as a novel nucleic acid detection technique that boasts of rapid detection speed, exceptional specificity, and remarkable sensitivity. This technique has found extensive applications in the detection of viruses, bacteria, and parasites ( 16 – 18 ). The amplification of target gene fragments is primarily dependent on three essential enzymes: recombinase, single-stranded DNA-binding protein (SSB), and strand-substituted DNA polymerase.…”

Section: Introductionmentioning

confidence: 99%

Establishment of two assays based on reverse transcription recombinase-aided amplification technology for rapid detection of H5 subtype avian influenza virus

Li,

Shang,

Wang

et al. 2023

Microbiol Spectr

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The avian influenza virus (AIV), a member of the type A influenza virus group, is harbored by avian hosts. The highly pathogenic H5 subtype of AIV has inflicted significant financial damage on the poultry industry and also presents a possible pandemic hazard to the worldwide human population. There is an urgent need for a detection method that is both rapid and accurate in identifying H5-AIV. In this study, two assays based on reverse transcription recombinase-aided amplification technology (RT-RAA) have been developed to detect H5-AIV, namely, real-time fluorescence and reverse transcription recombinase-aided amplification (RF-RT-RAA) and reverse transcription recombinase-aided amplification combined lateral flow dipstick (RT-RAA-LFD). The completion of the two methods can be achieved in 30 min, and there was no cross-reaction with the nucleic acid of other avian pathogens. The results indicated that the lowest detectable limit of RF-RT-RAA and RT-RAA-LFD for H5-AIV detection was 1 copy/μL, which was 100 times higher than that of RT-PCR (10 2 copies/μL). A total of 376 samples, consisting of 350 clinical and 26 experimental samples, underwent testing utilizing virus isolation, RF-RT-RAA and RT-RAA-LFD methods, respectively. The results demonstrated a high degree of consistency between those assays. In summary, both the RF-RT-RAA and RT-RAA-LFD methods demonstrated excellent specificity and sensitivity, making them ideal for use in both laboratory and field settings. IMPORTANCE Avian influenza virus (AIV) subtype H5 is a highly contagious zoonotic disease and a serious threat to the farming industry and public health. Traditional detection methods, including virus isolation and real-time PCR, require tertiary biological laboratories and are time-consuming and complex to perform, making it difficult to rapidly diagnose H5 subtype avian influenza viruses. In this study, we successfully developed two methods, namely, RF-RT-RAA and RT-RAA-LFD, for rapid detection of H5-AIV. The assays are characterized by their high specificity, sensitivity, and user-friendliness. Moreover, the results of the reaction can be visually assessed, which are suitable for both laboratory testing and grassroots farm screening for H5-AIV.

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The Development of a Real-Time Recombinase-Aid Amplification Assay for Rapid Detection of African Swine Fever Virus

Cited by 5 publications

References 28 publications

Vaccines for African swine fever: an update

Vaccines for African swine fever: an update

Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022

Establishment of two assays based on reverse transcription recombinase-aided amplification technology for rapid detection of H5 subtype avian influenza virus

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