Preliminary Establishment And Application Of Quantitative PCR Methods Chlamydial TaqMan-MGB Probe Multiple Fluorescence

Chlamydiosis is a significant nature infection disease, and it is mainly due to a variety of Chlamydia infection of mammals and birds and other animals. The disease is endemic, and Often suffered great damage and economic loss.At present,the detection methods of Chlamydia include cell culture method, indirect hem agglutination test (IHA), direct dyeing observation method and other methods.But the above methods for fast detection of Chlamydia still have many limitations. For example,cell culture method is always polluted by Mycoplasma and its sensitivity is relatively low. IHA is easy to operate, but differences between batches is large and positive detection rate is low. Direct dyeing observation sensitivity is low. Therefore, we need to establish a rapid, simple, specific, sensitive, stability, good detection method, and we apply this method to the early diagnosis of Chlamydia, epidemic monitoring and epidemiological investigation, etc.In order to ensure the healthy development of animal husbandry and speed up the pace of the modern animal husbandry forward, this study establishes multiple fluorescence quantitative polymerase chain reaction (PCR) based on Fluorescence quantitative PCR and this method can simultaneously detect three Animal Chlamydia TaqMan-MGB probe in a signal tube. This method has strong specificity, high sensitivity,as well as the characteristics of high stability. The main contents of the research include the following aspects:1、n comparison with the major outer membrane protein sequences of the nine kinds of Chlamydia in GenBank,We have designed three pairs of specific primers, three probes and1 for universal primers by using Primer Express3.0biology software.We amplified universal primers and built positive plasmid. Then we compared the results of the positive plasmid and the major outer membrane protein sequence have published in GenBank. The results show that the similarity between the constructed possitive plasmid and the outer membrane protein sequence published in GenBank, reached99%,97%and97%, respectively.2、By optimizing Single fluorescent quantitative PCR the concentration of the primer, the concentration of the probe and the Tm values, we established single fluorescent PCR detection method of the three Chlamydia..At the same time, we test the specificity and sensitivity of the method and set up a corresponding standard curve. The result show that this method can only amplify the purpose gene and can not specific amplify other Chlamydia. Ch. abortus minimum detectable amount of1.6copies/μL; Ch.psittaci single fluorescent quantitative PCR method detectable amount of1.6copies/μL, while the minimum detectable amount of Ch. pecorum is2.8copies/μL. The amplification efficiency of and standard curve equation and correlation coefficient are Ch.abortus R2=0.9999、amplification efficiency of105.02%,Y=-3.2075x+39.051;Ch.psittaci R2=0.999, amplification efficiency of101.7%、Y=-3.2815x+41.396;Ch.ecorum R2=0.9991、 amplification efficiency of109%、Y=-3.1245x+35.752;Thus three kinds of Chlamydia single fluorescent quantitative PCR has high specificity and sensitivity.3、Based on the established three kinds of Chlamydia single fluorescent quantitative PCR and by optimizing single fluorescent quantitative PCR primers concentration in the reaction system, the concentration of the probe, the Tm value temperature, we established the single pipe three Chlamydia at the same time of multiple fluorescent quantitative PCR detection method.The specificity test of Multiple fluorescent quantitative PCR detection method shows that:this method can specific amplification three segments of the purpose of Chlamydia, and for other Chlamydia without obvious amplification; The sensitivity test showed that:the lowest detectable amount of Ch.abortus is2.5x10copies/μL, Ch.ecorum is3.08copies/μL, and Ch.psittaci is1.6x10copies/μL. The standard curve equation, amplification efficiency, correlation coefficient R2, respectively are:Ch.abortus R2=0.999、amplification efficiency of103.2%、Y=-3.242x+38.813; Ch.psittaci R2=0.999、amplification efficiency of103.2%、Y=-3.24x+39.9; Ch.pecorum R2=0.999、amplification efficiency of96.4%、Y=-3.41x+39.325.Thus, this study established the Chlamydia multiple fluorescent quantitative PCR detection method has better properties.4、Using multiple fluorescent quantitative PCR method, and the detection method of OIE recommendations and animal Chlamydia shell polymerase chain reaction (PCR) test660pigs from Chongqing region nasal swabs, and960pig vaginal swabs.The positive of OIE recommends PCR detection method of test results were:20.9%,25.8%;Animal Chlamydia shell PCR detection method positive rate were:19.5%,24.9%.This study has established multiple fluorescent quantitative polymerase chain reaction (PCR) detected positive rate were:20.3%,25.6%.The result show that compared to detection method of OIE recommendations, the positive rate of this study has established multiple fluorescent quantitative PCR method less than0.8%;and more than1.5%for animal Chlamydia shell polymerase chain reaction (PCR). The established methods in our study can simultaneously the three Chlamydia while the other two methods cannot identify at the same time.In conclusion, this established multiple fluorescent quantitative PCR detection method can rapid differential diagnosis for single or mixed infection of Chlamydia samples.Not only for the clinical detection of Chlamydia provides scientific theoretical basis and technical support, and epidemiological investigation of Chlamydia and prevention and control is of great importance.