Establishment And Application Of TaqMan MGB Real-time PCR Assay Based On The Big Frog Virus 49L ORF

As the largest existing rare amphibians, Chinese giant salamander (Andrias davidianus) has very high economic values. Thus, its cultures develop rapidly in southwest and northwest regions in China. However, with the rapid expansion of farming scale, artificial temperature control and high density cultivation changed or even destroyed its natural life habits, causing the decline of its disease-resistant ability. Hence, varied diseases hit it continuously and cause serious influence to Chinese giant salamander breeding industry. Chinese giant salamander ranavirus (CGSRV) is one of a new Iridoviridae family virus found to cause serious damage of Chinese giant salamander health recently. It can lead to body surface ulcer, vast hemorrhage spot, head and limbs swelling and other symptoms. Moreover, it possesses rapid transmission, acute occurrence, high morbidity and high mortality. Therefore, it is also referred to as "the cancer of Chinese giant salamander". To better protect the Chinese giant salamander breeding industry, reduce the economic loss, the early and accurate diagnosis of CGSRV is very important for early warning. In this study, we hope to establish a TaqMan MGB real-time PCR detection technology, providing important technical methods to the early diagnosis and epidemiological investigation of Chinese giant salamander.A pair of primers according to 49L ORF which called US22 family gene of Chinese giant salamander ranavirus (CGSRV) was designed for PCR amplification to obtain the target gene sequence. The purified PCR product was subcloned into pMD19-T simple vector transformed into E. coli DH5a, designated pMD19-T-US22. The positive plasmid was extracted. The concentration of recombinant plasmid was measured, and then it was as a standard template with 10 times the gradient dilution. The specific primers and TaqMan MGB probe were designed for TaqMan MGB real-time PCR amplification, the reaction process was optimized, the standard curve was draw, and the TaqMan MGB real-time PCR detection method of 49L ORF of Chinese giant salamander ranavirus was established. Meanwhile, the specificity, sensitivity and repeatability of this technology were assessed and the unknown salamander blood samples were detected using this method. The results showed that US22 family gene sequence includes 639bp and it fits to the target sequences according to NCBI-Blast and Genbank. The standard curve was draw with an excellent linear correlation with a R2 value of 0.997 and the amplification efficiency of 99.14% after confirming the optimal annealing temperature of TaqMan MGB real-time PCR being 60 ?. The established method was highly specific for amplification of CGSRV, without cross-reactions with Cloudy pointed pond carp virus, Lymphocystis disease virus, Cyprinid herpesvirus ?, Aeromonas hydrophila, Aeromonas veronii, Aeromonas salmonicida, Pseudomonas fluorescens and Edwardsiella tarda. Its sensitivity is nice and a minimum of 2.00×101copies/?L of standards couldbe detected, which indicated that the sensitivity of real-time PCR is about 3 orders of magnitude gradient than that of the conventional PCR test (the conventional PCR test detecting a concentration with 2.00×104copies/?L). The standard deviation and coefficients of variance (CV) were both less than 2%, which indicated a decent reliability. In the detection on 73 suspected salamander blood samples, TaqMan MGB quantitative PCR could detect 45 positive samples with a relevance ratio of 61.64%, while PCR could detect 32 positive samples corresponding to the results of TaqMan MGB quantitative PCR. Comparing with conventional PCR, TaqMan MGB qPCR detection coincidence rate (Po value) of 82.19%, and Kappa value of 0.6537, and both possess good consistency. However, TaqMan MGB real-time PCR could detect lower concentration of positive infection than that of the traditional PCR method, showing better sensibility than PCR.Owing to the TaqMan MGB real-time PCR detection technology established in this studyaccording to 49L ORF of Chinese giant salamander ranavirus allowing for a rapid, specific, sensitive, repeatable, quantitative detection, Suitable for testing a large number of samples and implement live body by a blood test, it will be useful for early detection of CGSRV.