The quantitative characteristics of infection with infectious precocity virus (IPV) revealed with a new TaqMan probe-based real-time RT-PCR method

Infectious precocity virus (IPV) is a recently reported RNA virus associated with the sexual precocity of Macrobrachium rosenbergii, so-called iron prawn syndrome (IPS), and has caused huge economic losses in the prawn farming industry. We developed a semiquantitative approach for diagnosing IPS based on clinical signs and established a one-step, TaqMan probe-based reverse-transcription quantitative PCR (TaqMan-RT-qPCR) method to detect and quantify IPV. The detection limit of this method was as low as 1.00 × 100 copy/reaction. The standard curve between 1.00 × 100 to 1.00 × 109 copies/reaction showed a high correlation coefficient (R2 = 0.999). Quantitative detection of different tissues in M. rosenbergii infected with IPV revealed the characteristic neurotropism of the virus with the highest loads in eyestalks and the brain. The assay showed that the risk of IPS in a farm might be significant if its prawn samples have a load of IPV copies above 103 copies/μg-RNA. Most symptomatic cases with typical IPS clinical signs had a high load of IPV above 3 × 105 copies/μg-RNA in eyestalk or 2 × 103 copies/μg-RNA in cephalothorax tissues. The results showed that TaqMan-RT-qPCR was about 13 and 1300 times more sensitive than reported nested RT-PCR and first-step RT-PCR. Bayesian estimation based on Markov Chain Monte Carlo (MCMC) showed that the TaqMan-RT-qPCR method has the highest diagnostic sensitivity (DSe), and the clinical diagnosis method has the highest diagnostic specificity (DSp). This study developed a specified clinical diagnosis method for IPS and a rapid and sensitive method for IPV detection in farmed prawns and revealed the quantitative characteristics of infection with IPV in M. rosenbergii and provided quantitative statistical evidence supporting IPV being the causative agent of IPS.