Development of a fluorescent quantitative polymerase chain reaction technique for detection of grass carp reovirus HZ08 strain

Liu, Bao-Qin; WeiWei, Zeng; Wang, Qing; Zhang, Lesheng; Wang, Yingying; Shi, Chao; Shu-qin, WU

doi:10.3724/sp.j.1118.2012.00329

Cited by 4 publications

(5 citation statements)

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“…Vaccination and early diagnosis is the efficient way for preventing and controlling GCHD (Brudeseth et al, 2013). Numerous techniques have been documented for identifying GCRV infection, such as electron microscopy, virus isolation, nucleic acid sequence amplification plus ELISA (NASA-ELISA), LAMP, and RT-PCR (Liu et al, 2012;Zeng et al, 2013;Wang et al, 2020). Nonetheless, the above technologies are timeconsuming, labor-intensive, nonsensitive, impossible for quantification and need AGE of amplified product.…”

Section: Discussionmentioning

confidence: 99%

Rapid detection of grass carp reovirus type 1 using RPA-based test strips combined with CRISPR Cas13a system

Li,

Cao,

Chen

et al. 2023

Front. Microbiol.

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IntroductionDue to the existence of grass carp reovirus (GCRV), grass carp hemorrhagic disease occurs frequently, and its high pathogenicity and infectivity are great challenges to the aquaculture industry. As a highly pathogenic pathogen, the outbreak of hemorrhagic disease often causes tremendous economic losses. Therefore, it is important to rapidly and accurately detect GCRV on site to control timely.MethodsIn this study, recombinant enzyme amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system was employed to establish a method to detect the vp7 gene of grass carp reovirus type 1. This method can be adopted for judging the results by collecting fluorescence signal, ultraviolet excitation visual fluorescence and test strip.ResultsCombined with the RPA amplification experiment, the detection limit of the RPA-CRISPR method can reach 7.2 × 101 copies/μL of vp7 gene per reaction, and the detection process can be completed within 1 h. In addition, this method had no cross-reaction with the other 11 common aquatic pathogens. Then, the performance of the RPA-CRISPR/Cas13a detection method was evaluated by comparing it with the real-time fluorescence quantitative PCR detection method of clinical samples. The results of RPA-CRISPR/Cas13a detection were shown to be in consistence with the results obtained from the real-time fluorescence quantitative PCR detection. The coincidence rate of this method with 26 GCRV clinical samples was 92.31%.DiscussionIn summary, this method has high sensitivity, specificity and on-site practicability for detecting GCRV type 1, and has great application potential in on-site GCRV monitoring.

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Section: Discussionmentioning

confidence: 99%

Rapid detection of grass carp reovirus type 1 using RPA-based test strips combined with CRISPR Cas13a system

Li,

Cao,

Chen

et al. 2023

Front. Microbiol.

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“…Thus, the GCRV HZ08 in fishes can be identified during the early stages of infection and the disease can be effectively controlled before the infection becomes severe. In a previous study, a real-time PCR was developed with a detection limit of six copies of viral genomic RNA (Liu et al ., 2012). Although the RT-LAMP assay is slightly less sensitive than real-time PCR, it is still considered to be superior as it is a comparatively simple and rapid method which can be performed both in the field and in the laboratory.…”

Section: Discussionmentioning

confidence: 99%

“…In order to test the sensitivity, RNA standards of known concentrations were prepared by fluorescent quantitative PCR technique as previously described (Liu et al ., 2012). A 10 fold series of dilutions spanning 1-10 5 copies per tube was used as template to test the sensitivity of RT-LAMP and RT-PCR for detection of GCRV HZ08.…”

Section: S E N S I T I V I T Y O F Rt -L a M P A N D Rt -P C Rmentioning

confidence: 99%

“…Because GCRV HZ08 induced no cytopathic effects in host cell and no serological tests were available, conventional reverse‐transcription polymerase chain reaction (RT‐PCR) and real‐time RT‐PCR assays based on detection of the segment 6 (s6) gene were developed for primary diagnosis of GCRV HZ08 infection. Even these assays had good sensitivity and specificity (Liu et al ., ), they were time‐consuming and laborious, requiring centralized laboratory facilities and clinical specimen submissions. The assays usually resulted in the delay of GCRV diagnosis.…”

Section: Introductionmentioning

confidence: 99%

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A one‐step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 strain

Zeng

Wang

et al. 2013

Journal of Fish Biology

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Six reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primers designed against conserved regions of segment 6 (s6) gene were used for the detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 subtype. The entire amplification could be completed within 40 min at 62·3° C. The RT-LAMP showed higher sensitivity than reverse-transcription polymerase chain reaction (RT-PCR). The RNA detection limit was 10 copies µl⁻¹ for RT-LAMP assay and 100 copies µl⁻¹ for conventional RT-PCR. In specificity tests, no cross-reactivity was detected in other viruses from common aquatic animals. In addition, the reaction results can be visualized by using calcein fluorescent dye. Furthermore, a total of 86 samples were tested by RT-LAMP, RT-PCR and virus isolation. The results demonstrated that all 54 specimens identified as positive by virus isolation were also positive when detected by RT-LAMP. Seven out of 54 samples, however, were misidentified by RT-PCR. The RT-LAMP method is more accurate than conventional RT-PCR. The results indicate that RT-LAMP has potential as a simple and rapid diagnosis technique for the detection of GCRV HZ08 subtype infection.

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“…Grass carp were kindly provided by a fish farm located in Guangzhou (Guangdong, China) with a mean total length and body weight of 20.0 ± 0.5 cm and 100.0 ± 0.5 g, respectively (data are presented as mean values ± SD). Possible viral contamination of the fish but also of the used food was checked by reverse transcription quantitative real‐time PCR (RT‐qPCR) to verify that they were free from different pathogens (Liu et al., ). Animals welfare procedures were performed in compliance with the guidelines of the Animal Experiment Committee, South China Agricultural University.…”

Section: Methodsmentioning

confidence: 99%

Development of a VP38 recombinant protein‐based indirect ELISA for detection of antibodies against grass carp reovirus genotype II (iELISA for detection of antibodies against GCRV II)

Zeng

Wang

Guo

et al. 2018

Journal of Fish Diseases

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Currently, serological assays for grass carp reovirus genotype II (GCRV‐II) diagnosis are not available. In this study, an indirect enzyme‐linked immunosorbent assay (ELISA) for the detection of antibodies against GCRV‐II was developed. The structural protein VP38 of GCRV‐II was used as the coating antigen. Monoclonal antibodies (mAb) against IgM of grass carp labelled with HRP were used as a secondary antibody. The antigen concentration and serum dilution were optimized using chess board titration. Furthermore, the specificity of indirect ELISA assay was confirmed by cross check with sera positive for other grass carp pathogens. In comparison with results obtained from indirect immunofluorescence assay (IFA) and Western blot by testing of 60 serum samples to evaluate the sensitivity and specificity of the ELISA, agreement between 90% and 96.7% was reached, respectively. A serological survey was performed using the assay with grass carp field serum samples. The seropositive rate of the 242 serum samples was 69.8%. In conclusion, the developed indirect ELISA is a very specific and sensitive test that will be useful for large‐scale serological surveys to detect indirectly GCRV II infections as well as to monitor the changes of antibody level after immunization.

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Development of a fluorescent quantitative polymerase chain reaction technique for detection of grass carp reovirus HZ08 strain

Cited by 4 publications

References 4 publications

Rapid detection of grass carp reovirus type 1 using RPA-based test strips combined with CRISPR Cas13a system

Rapid detection of grass carp reovirus type 1 using RPA-based test strips combined with CRISPR Cas13a system

A one‐step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 strain

Development of a VP38 recombinant protein‐based indirect ELISA for detection of antibodies against grass carp reovirus genotype II (iELISA for detection of antibodies against GCRV II)

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