Research Of Molecular Methods For The Detection Of Animal Chlamydial Pathogens And The Development And Application Of PCR Diagnostic Kit

Chlamydiosis is a very important zooanthroponotic disease in humans and in someanimal species such as mammals，birds and insects. Chlamydiaceae are Gram-negativeobligate intracellular bacteria. According to a newly proposed classification,the familyChlamydiaceae is divided into two genera:Chlamydia including the species Chlamydiatrachomatis(CT), Chlamydia muridarum(C.muridarum), Chlamydia suis(C.suis)andChlamydophila including the species Chlamydophila Pneumoniae (Cpn),Chlamydophila pecorum(Ch.pecorum), Chlamydophila psittaci(Ch.psittaci),Chlamydophila abortus(Ch. abortus), Chlamydophila caviae(Ch. caviae) andChlamydophila felis(Ch.felis). They are responsible for a broad range of diseases inanimals and humans. CT, Cpn and Ch. psittaci infect both human and variety kinds ofanimals，Ch. pecorum, Ch. abortus and C. suis mainly infect some demostic and wildanimals. Therefore，animal chlamydial infection has a very important public health andeconomical importance. Rapid and accurate detection methods of these pathogens arenecessary for the control and prevention of this disease.The aim of the current study was to develop a nested multiplex polymerase chainreaction (nmPCR) assay and a diagnostic kit to simultaneously detect the3chlamydialpathogens (include Cpn，Ch. abortus and C. suis)in clinical samples，to develop a realtime-PCR assay and a LAMP assay for the family Chlamydiaceae. The nmPCR kit wasapplied to investigate chlamydial infection in sheep, goat and pigs.Results of the current study as follows:(1) A family-specific PCR was established to detect pathogens of familyChlamydiaceae. Forthermore, an nmPCR for differential identification of C. suis，Ch.abortus and Cpn was also developed to simultaneously detect the three chlamydialpathogens. In the first round of the nmPCR, one pair of family specific primer was usedto amplify the1100bp fragment of chlamydial ompA gene. In the second round of thenmPCR, inner primers were designed for Ch. abortus, C. suis and Cpn. Each pathogenproduced a specific amplicon with a size of340bp,526bp and267bp respectively. Theassay was sensitive and specific for detecting target pathogens in both cell cultures andclinical specimens. These results, incorporated with the improved rapid DNA extractionprotocol, suggest that the nmPCR could be a promising assay for differentialidentification of chlamydial strains of porcine，sheep and goat. (2) A nmPCR diagnostic kit was developed based on above mentioned research. Itgoes through stable and consistent experiments with good realiabilty and repeatability.The sensitivity of the two-step nmPCR assay developed here was under5IFU.mL-1and10times more sensitive than that of the16S rDNA nPCR and23S rDNA real-timePCR assays.(3) A realtime-PCR based on ompA gene was established to detect Chlamydiaceae.This method can detect target DNA as low as200copies/μL and could be applied todiagnosis of Chlamydiaceae infection.(4) A loop-mediated isothermal amplification method (LAMP) based on ompAgene was set up to rapidly detect pathogens of family Chlamydiaceae. The amplificationefficiency of the LAMP method is extremely high because there is no time loss forthermal change, since the reaction is isothermal.Positive result of LAMP showsyellow-green color and be easily read when Calcein is added into the reaction mixture.Thus, this method can be used as a rapid screen assay for pathogens of familyChlamydiaceae. Positive result should be confrmed using other methods such asreal-time PCR assays avoiding false positive reaction.(5) The nmPCR kits were applied to investigate chlamydial infection in sheep andgoat in a farm in QingHai province and pigs in ChongQing municipality, China. This isthe first time that the infection of C.suis and Cpn was found in swabs of sheep, goat andpigs in China.