Establishment and preliminary application of a nested RTPCR for the detection of Macrobrachium rosenbergii nodavirus

Abstract:

Macrobrachium rosenbergii nodavirus (MrNV) was the main pathogen of white tail disease (WTD), which was defined as the notifiable disease of aquatic animals by Office International des Epizooties (OIE). The purpose of this study is to develop a nested RT-PCR method for the detection of MrNV. Two pairs of specific primer were designed and synthesized according to the published MrNV capsid protein gene sequence in GenBank. After the nested RTPCR method was optimized, the specificity and sensitivity were assayed. This method can produce 205 bp amplicons of MrNV capsid protein gene with the following conditions: 0.8 μmol/L optimal concentration of primer, 56 ℃ anneal temperature and 2.0 mmol/L Mg²⁺ concentration, and had no crossreaction with three other prawn pathogens such as taura syndrome virus (TSV), white spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV). The results of sensitivity test showed that the nested RT-PCR was 10³ times more sensitive than that of onestep RTPCR, and the minimum detection limits of the nested RTPCR were 2.612×10^-2 fg MrNV RNA.108 clinical Macrobrachium rosenbergii samples from Guangxi were tested by the two RT-PCR, and 9 out of 108 samples were positive by nested RT-PCR, while 3 out of 108 samples by onestep RT-PCR, which showed that the nested RT-PCR can boost positive detection rate of MrNV. The results indicated that the nested RT-PCR method for the detection of MrNV was successfully established and this method has high specificity and sensitivity, and could be used for the early diagnosis of acute and latent infections of MrNV. This study provides a reliable way for clinical detection, epidemiological investigation, import and export quarantine and laboratory studies on MrNV.