Establishment Of Real-time Quantitative PCR And ELISA For Detection Of Chlamydia Abortus

Chlamydia abortus（C.abortus）is the main pathogen causes enzootic abortion of ewes（EAE）,which has a wide range of distribution all over the world,and can mainly cause large-scale abortion in ruminants.Infected animals will become new reservoirs,and women who are pregnant have prolonged contact with the source of infection may trigger severe abdominal pain,inflammation or respiratory problems,which can lead to miscarriage,premature delivery,or stillbirth.C.abortus,an important pathogen of zoonotic diseases,poses a huge threat to the sustainable development of animal husbandry and human health worldwide.It is of great significance to research on specific,sensitive and rapid diagnostic methods for the prevention,control and epidemiological investigation of the disease.Therefore,in this study,we established a real-time PCR diagnostic method based on the Chlamydial protein-like activity factor（CPAF）gene and an indirect ELISA diagnostic method based on recombinant protein CPAF to detect CPAF antibodies.The specific research results are as follows:1.A real-time fluorescence quantitative PCR method for C.abortus was established based on the gene encoded by CPAF of C.abortus.In this study,a set of specific primers and probe for C.abortus were designed based on the nucleotide sequences of CPAF of different Chlamydia spp.published by Gen Bank,and the standard curve of this method was built using a template which is the constructed recombinant plasmid p MD19T-CPAF.The minimum detection concentration of the recombinant plasmid in this method is 26copies/μL,which is 10 times sensitive compared to the ordinary PCR method.The real-time fluorescent quantitative PCR method founded in this study has no cross-reactivity with other pathogens that can induce similar symptoms.Both the intra-group and inter-group variable coefficient are less than 3%.Through the test of clinical samples,the method established in this study is suitable for large-scale clinical sample detection and has a higher sensitivity than the fluorescence quantitative PCR method,which is recommended by the World Organization for Animal Health（OIE）.2.An indirect ELISA method for detecting CPAF antibodies based on the recombinant Chlamydia abortus protein CPAF is established.In this study,the purified recombinant protein CPAF was used to coat the ELISA plate.The optimal reaction conditions after optimization are followed:the coating conditions are 0.25μg/wells,4 ℃,16 h,37 ℃ and blocking for 30 minutes,and the serum to be tested is diluted at a ratio of 1:300,incubation at 37 ℃ for 60 minutes,and the enzyme-labeled secondary antibody is diluted at a ratio of1:30000,incubating at 37 ℃ for 45 minutes and colored at 37 ℃ for 10 minutes.The clinical serum sample will be classified as positive when the value of OD₄₅₀ is higher than0.442;and the minimum detection limit is 1:2560.the ELISA plate can be stored at 4 ℃,﹣20 ℃ or﹣70 ℃ for at least 3 months and the coefficient of variation of the repeatability test within and between groups are less than 8%.The detection results of clinical samples showed that the method established in this study was more sensitive than indirect hemagglutination assay（IHA）,and could determine the negative and positive results of"suspicious"samples in IHA detection results.The positive rate of detection was consistent with the detection rate of imported kits abroad,but the cost of this method was lower,indicating that the method established in this study is more suitable for the clinical large-scale detection of Chlamydia abortus serology in China.In summary,a real-time fluorescence quantitative PCR and indirect ELISA method for the detection of C.abortus was established in this study.Both methods have the advantages of high sensitivity and good repeatability,and can be applied in the clinical diagnosis of C.abortus,and are expected to be developed into the corresponding detection kit.Thus,technical support can be provided by the methods in this study for the high-throughput detection and epidemiological investigation of abortion chlamydiosis,which can make feasible technical means for the formulation of comprehensive prevention and control measures for abortion chlamydiosis.