Establishment And Application Of A Detection Method Of Lawsonia Intracellularis Through Taq Man Fluorescence Quantitative PCR

Porcine Proliferative Enteritis（PPE）is a porcine contagious disease caused by Lawsonia Intracellularis（LI）.It is characterized by nodular proliferation of immature intestinal cells in the ileum and colonic crypts.L.Intracellularis is an obligate intracellular Gram-negative bacterium.It has curve,comma or S-shaped bacillus with tapered or blunt ends.The bacterium is 1.25～1.75μm long and 0.25～0.43μm wide,compacted in a trilaminar corrugated outer membrane,without fimbriae and spores.The bacteria are strictly intracellular parasitism,and the optimal culture conditions are37°C,82.2%N₂,8.8%CO₂,and 6～8%O₂ in a microaerophilic environment.LI has a wide range of hosts,pig is one of the major host;meanwhile,it also can infect horse,hamster,rabbit,goat,deer,ostrich,raccoon,coyote,macaque,squirrel,etc.PPE has a wide geographic distribution,almost covering the whole world.Furthermore,subclinical symptoms are the most common in PPE,and most of infected pigs do not show obvious clinical symptoms.This study focuses on screening the primers and probes with high sensitivity and stable amplification by SYBR GreenⅠand Taq Man fluorescence quantitative PCR（FQ-PCR）method.On this basis,the LI detection method based on Taq Man fluorescent quantitative PCR was established and successfully adapted to detecting the LI pathogens in pig feces in order to investigating PPE infection rate in the pig farms.1.Establishment of Taq Man FQ-PCR methodThe types of microorganisms in pigs’intestine are complex and diverse.There are about 400～500 species of microbes in each gram of intestinal contents,and more than80%of the bacterial species are unknown species.So far,the regular methods for detecting bacteria in the intestinal tract of pigs have high false positive results.Thus,fluorescent quantitative PCR,which has high specificity and sensitivity,could be a good method for detecting bacteria in intestine.In this study,based on the LI whole genome sequence with the accession number AM180252 in Gen Bank,50 pairs of primers were designed for 36 reading frames.These primers are screened by SYBR Green I FQ-PCR.the results showed that the melting curve peaks varied considerably.These primers could be divided into 3 categories according to the amplification results:1)The primers without target fragments（13 pairs）;2)The primers with primer dimers or（and）non-specific fragments（34 pairs）;3)The primers with unique target fragments（3 pairs）,LI-0662q PCR-F1/R1,LI-0897q PCR-F1/R1,LI-0352q PCR-F1/R1.For the primers with unique target fragments,the corresponding three probes and27 pairs of primers surrounding the probe were designed and further verified by the Taq Man FQ-PCR.According to the amplification efficiency,27 pairs of primers were sorted,and the primer with the highest amplification efficiency in both cases was screened out.In this study,a pair of primers（LI-0662-q PCR-F3/R1）and the corresponding probe（LI-0662-q PCR-P）targeting to the LI0662 reading frame were selected as the best candidate primers for the establishing the LI FQ-PCR detection method.After optimizing the reaction system and reaction conditions,the FQ-PCR detection assay was performed on the nucleic acid samples of each dilution of the standard quality pellets,and the results showed the standard curve equation wasy=-3.4085 lg x+39.671（x:copy number,y:Ct value）,and the correlation coefficient R²is 0.9981;there was a good linear relationship between the Ct value and different concentration templates（2.95×101～2.95×106 copies/（?）L）.The limitation detection could reach 5 copies/μL and the repeatability test shows the coefficient of variations（CV）were less than 1%.Different amounts of live ileitis vaccine bacteria were added to DMEM and negative feces respectively,then detected through the method established in this study.The results showed that the recovery rate of LI in DMEM and fecal samples were 85.00～100%and 4.27～20.85%,respectively.Therefore,the LI Taq Man FQ-PCR method established in this study can meet the requirements of rapid,quantitative,accurate and sensitive detection of Lawsonia Intracellularis in pig feces or tissue samples.2.The clinical application of Taq Man FQ-PCR methodFour fecal samples were collected from sows with diarrhea,but without bad mental and appetite conditions in a pig farm in Jiangxi Province.Through the LI Taq Man FQ-PCR assays,all samples showed positive results.The fecal DNA No.3（Ct 27.33）was amplified and sequenced with detection primer LI1022-F/R（581bp）of LI1022 reading frame.The result showed that the sequence belongs to the LI,and has homology with three LI sequences:NC-008011.1（100%）,CP004029.1（100%）,EU621794.1（99.82%）.From 2020 to 2021,383 fecal samples from 12 pig farms in Jiangxi Province were collected and detected the LI pathogen through the Taq Man FQ-PCR detection method.The results showed that there were 10 out of 12 farms were LI-positive（the positive rate was 83.33%）and 109 out of 383 fecal samples were LI-positive（the positive rate was 28.46%）,of which 28 were LI-positive in nursery pigs（positive rate of 36.36%）,4were LI-positive in fattening pigs（positive rate of 14.29%）,25 were LI-positive in reserve pigs（positive rate of 23.36%）,and 52 were LI-positive in sows（positive rate of 28.73%）.