The Development Of Real Time Quantitative PCR In The Detection Of Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae is the causative agent of mycoplasmal pneumonia of swine (MPS), meanwhile, it is an important causative agent of Porcine Respiratory Disease Complex (PRDC). The main clinical symptom is characterized by slow growth and decreased feed efficiency, which bring the serious economic loss to swine industry.The classical methods for quantitative detection of Mhp include the CCU(color change unit), CFU (colony forming unit) and nephelometry assay. The error of the characterization for Mhp is unavoidable since the above methods are time-consuming, labor-intensive and the casual contamination. In the present study, a fluorescent quantitative PCR assay with a TaqMan probe for Mhp detection was established, which provided the laboratory support for the reasearch of the growth characteristic and quantitation in culture. The primary contents and results are list as follows:1. The establishment of FQ-PCR for Mhp detection: A pair of primers together with a TaqMan probe were designed according to the published sequence of Mhp (p110 gene) listed in GenBank. Meanwhile, the standard plasmid pEASY-T1-p110 containing partial sequence of p110 was constructed as a template.The optimization was carried out for FQ-PCR assay to detect the Mhp quantitation. The results showed the sensitivity to Mhp was 13copy/μL, and the linear equation was expressed as Ct=-3.320×lg(copy/μL)+32.545. No corss-reaction was found in other mycoplasma, virus and bacteria.The final measure value corresponding to the initiative DNA concentration of 1.3×10⁵, 1.3×10⁴, 1.3×10³ copy/μL was 1.332×10⁵, 1.293×10⁴ and 1.260×10³ copy/μL, respectively. And the p of u test was 0.5322, 0.8634 and 0.4872(p>0.05), which demeostrate the method posses the trait of high-sensitivity, good specificity and accuracy.2. The comparision among the three quantative methods: The growth curve of Mhp was investigated by CCU, nephelometry and FQ-PCR, and a primary regression analysis was carried out. The results showed FQ-PCR was high correlated with nephelometry, and the regression equation was OD=0.922398×lgcopy-6.578453. Within 54hrs, the results of FQ-PCR was high correlated with CCU with a regression equation depicted as lgCCU =2.501728×lgcopy-13.693792. Verification test of the regression equation indicated the difference of lgCCU/mL and OD₄₂₀ between the theoretical value and real value was not obvious. So, the CCU/mL and nephelometry may be deduced according to the results of FQ-PCR because of its applicability.