The present study was aimed to establish a novel TaqMan real-time PCR (RTm-PCR) for detecting and typing bovine viral diarrhea virus (BVDV), and also to develop a diagnostic proto- col which simplifies sample collection and processing. Universal primers and TaqMan-MGB probes were designed from the known sequences of conserved 5′ - and 3′-untranslated regions (5’UTR, 3’UTR) of the NADL strain of BVDV. Prior to optimizing the assay, cDNAs were tran- scribed in vitro to make standard curves. The sensitivity, specificity and stability (reproducibility) were evaluated. The RTm-PCR was tested on the 312 feces specimens collected from persistently infected (PI) calves. The results showed the optimum conditions for RTm-PCR were 17.0 μmol/L primer, 7.5 μmol/L probe and 51.4°C annealing temperature. The established TaqMan RTm-PCR assay could specially detect BVDV without detecting any other viruses. Its detection limit was 1.55×100 copies/μL for viral RNA. It was 10000-fold higher than conventional PCR with excel- lent specificity and reproducibility. 312 samples were tested using this method and universal PCR from six dairy farms, respectively. Positive detections were found in 49 and 44 feces samples, respectively. The occurrence rate was 89.80%. In conclusion, the established TaqMan RTm-PCR could rapidly detect BVDV and effectively identify PI cattle. The detection limit of RTm-PCR was 1.55 copies/μL. It will be beneficial for enhancing diagnosis and therapy efficacy and reduce losses in cattle farms.
Geomechnical model testing has been widely applied as a kind of research technique in underground engineering problems. However, during the practical application process, due to the influence of many factors, the desired results cannot be obtained. In order to solve this problem, based on the measurement requirements of the model test, combined with FBG(Fiber Bragg Grating) sensor technology and traditional measurement methods, an FBG monitoring system, Micro-multi-point displacement test system, resistance strain test system and surrounding rock pressure monitoring system are developed. Applying the systems to a model test of the tunnel construction process, the displacement in advance laws of tunnel face, radial displacement distribution laws and surrounding rock pressure laws are obtained. Test results show that a multivariate information monitoring system has the advantage of high precision, stability and strong anti-jamming capability. It lays a solid foundation for the real-time data monitoring of the tunnel construction process model test.