Establishment of Optimized Parallel Real-time PCR for the Diagnosis of Semen-Transmitted Swine Reproductive Diseases
Date Issued
2011
Date
2011
Author(s)
Chen, Yi-Ting
Abstract
The epidemiology of swine reproductive diseases has changed significantly following the introduction of artificial insemination (AI). The advantages of AI include prevention of disease transmission, breeding improvement, cost reduction, and overcoming the impediment in natural mating. However, it has also been shown more serious disease spreading and economic loss can occur due to incomplete pathogen inspection in the semen used for AI. Under the current high demanding of AI in pig industry, it is required that each semen sample used for AI needs to be thoroughly examined 3-4 hours before shipping to ensure its quality. Therefore, the speed for semen examination is critical. In the present study, a high throughput multiplexed, parallel real-time polymerase chain reaction (rt-PCR) was established to simultaneously detect 9 important reproductive pathogens, including pseudorabies virus (PRV), parvovirus (PPV), porcine circovirus type 2 (PCV2), Japanese encephalitis virus (JEV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Toxoplasma gondii, Leptospira spp., and Brucella suis, in swine semen. Reagents of Clontech® (Clontech laboratories, Inc., USA) and Mastercycler® ep realplex (Eppendorf, Germany) were used for the establishment of the parallel rt-PCR for the 9 pathogens, under the following conditions: initial denaturation at 95 ℃ for 30 sec, 45 cycles of denaturation at 95 ℃ for 5 sec, and annealing and extension at 60 ℃ for 30 sec of SYBR Adventage® qPCR premix; initial reverse transcription at 48 ℃for 20 min, followed by initial denaturation at 95 ℃ for 3 min, 45 cycles of denaturation at 95℃ for 15 sec, and annealing and extension at 60 ℃for 1 min of one-step qRT-PCR SYBR kit; furthermore, initial denaturation at 95 ℃for 3 min, 45 cycles of denaturation at 95 ℃ for 15 sec, and annealing and extension at 60 ℃ for 1 min of QTaq DNA polymerase mix; initial reverse transcription at 48 ℃for 20 min, followed by initial denaturation at 95 ℃ for 3 min, 45 cycles of denaturation at 95℃ for 15 sec, and annealing and extension at 60 ℃for 1 min of QTaq one-step qRT-PCR kit. Two fluorescence detection methods for monitoring rt-PCR were included. The dsDNA-binding dye SYBR Green I was used along with the analysis of the melting temperature (Tm) of the amplified products for primer validation and singleplex condition optimization. TaqMan probes were then designed so that they could anneal within a DNA region amplified by a specific set of primers to develop a rapid multiplexed, parallel rt-PCR for the detection of the 9 above mentioned pathogens simultaneously. Furthermore, analysis of the specificity and sensitivity to validate the stability and reproducibility of each assay was performed. The optimal detection limit for SYBR Green was 17.6 copies/μL for PPV, 19.6 copies/μL for Leptospira spp., and 101~2copies/μL for others. The optimal detection limit for TaqMan was 3.6 copies/μL for JEV and 101copies/μL for others. The established multiplexed parallel rt-PCR could check the 9 target pathogens simultaneously and rapidly which could ensure the completion of quality examination of semen within 3-4 hours and prevent the spread of reproductive pathogens via semen in the future. The improved semen quality control for AI will effectively enhance the reproduction performance in pig industry and reduce the associated economic loss.
Subjects
parallel real-time polymerase chain reaction
pig reproductive disorders
arti?cial insemination
Type
thesis
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