动物衣原体分子检测方法研究和诊断试剂盒的研制及应用
摘要
衣原体病是由各类衣原体感染人类、哺乳动物、禽类以及节肢昆虫所引起的一类重要疾病，有些衣原体病为人畜共患传染病。衣原体革兰氏染色阴性，严格细胞内寄生。目前，衣原体科分为衣原体和嗜衣原体两个属。衣原体属包括沙眼衣原体、鼠衣原体、猪衣原体；嗜衣原体属包括肺炎嗜衣原体、家畜嗜衣原体、鹦鹉热嗜衣原体、流产嗜衣原体、豚鼠嗜衣原体和猫嗜衣原体。衣原体引起人类和动物多种疾病，沙眼衣原体、肺炎嗜衣原体和鹦鹉热嗜衣原体感染人类及各种动物；家畜嗜衣原体、流产嗜衣原体和猪衣原体主要感染家畜和某些野生动物。因此，本病具有十分重要的公共卫生和经济意义，建立快速准确的鉴别诊断方法对衣原体病预防和控制尤为重要。
本论文基于衣原体的主要外膜蛋白基因（ompA）开发针对猪衣原体（C.suis)、流产嗜衣原体（Ch.abortus)及肺炎嗜衣原体(Cpn)的多重套式PCR（nested multiplexpolymerase chain reaction assay，nmPCR)检测方法及试剂盒，衣原体荧光定量PCR检测方法和LAMP检测方法。并应用nmPCR试剂盒对青海某羊场和重庆地区种猪场进行了衣原体病的流行病学研究。获得以下研究结果：
(1)本文建立了衣原体科特异PCR检测方法以及流产嗜衣原体、猪衣原体和肺炎嗜衣原体的单管同步分型的多重套式PCR（nmPCR）检测方法。在nmPCR首轮扩增中，可以扩增出衣原体科特异性的1100bp核酸片段。在此基础上，第二轮nmPCR扩增获得种特异性扩增片段：流产嗜衣原体340bp，猪衣原体526bp和肺炎嗜衣原体267bp。该方法具有灵敏度高和特异性的特点，与改良DNA提取方法相结合，可以有效实现对细胞培养物、猪、绵羊和山羊鼻咽拭子、粪拭子及组织样品中三种衣原体同时进行检测。
（2）本文研发了与猪衣原体、流产嗜衣原体及肺炎嗜衣原体的单管同步分型的多重套式PCR检测方法相匹配的标准化检测试剂。通过稳定性试验和实验室间验证试验，证明了该检测试剂盒具有良好稳定性和可重复性。
（3）本研究建立了衣原体的实时定量PCR检测方法，灵敏度高，特异性强。最低可以检测200拷贝/μL质粒DNA，可用于衣原体的诊断和流行病学调查。
（4）本文基于ompA基因，建立了衣原体科LAMP检测方法。该方法具有特异性强、灵敏度高及实验条件要求较低的特点，可以在水浴锅中完成，在添加了钙黄绿素后，阳性的LAMP扩增反应呈现明显的黄绿色，使检测结果的非常直观，容易判读。本法适宜于临床上对衣原体病进行现场快速筛查，但阳性样品可用其他方法进行分型确证。
（5）应用本文研发的猪衣原体、流产嗜衣原体及肺炎嗜衣原体的单管同步分型的多重套式PCR检测试剂盒对青海羊场和重庆猪场采集的样品进行检测。首次从猪检测到C.suis和Cpn，首次从我国绵羊和山羊检出C.suis和Cpn，并发现C.suis、Cpn和Ch.abortus对绵羊和山羊的混合感染。
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.
本论文基于衣原体的主要外膜蛋白基因（ompA）开发针对猪衣原体（C.suis)、流产嗜衣原体（Ch.abortus)及肺炎嗜衣原体(Cpn)的多重套式PCR（nested multiplexpolymerase chain reaction assay，nmPCR)检测方法及试剂盒，衣原体荧光定量PCR检测方法和LAMP检测方法。并应用nmPCR试剂盒对青海某羊场和重庆地区种猪场进行了衣原体病的流行病学研究。获得以下研究结果：
(1)本文建立了衣原体科特异PCR检测方法以及流产嗜衣原体、猪衣原体和肺炎嗜衣原体的单管同步分型的多重套式PCR（nmPCR）检测方法。在nmPCR首轮扩增中，可以扩增出衣原体科特异性的1100bp核酸片段。在此基础上，第二轮nmPCR扩增获得种特异性扩增片段：流产嗜衣原体340bp，猪衣原体526bp和肺炎嗜衣原体267bp。该方法具有灵敏度高和特异性的特点，与改良DNA提取方法相结合，可以有效实现对细胞培养物、猪、绵羊和山羊鼻咽拭子、粪拭子及组织样品中三种衣原体同时进行检测。
（2）本文研发了与猪衣原体、流产嗜衣原体及肺炎嗜衣原体的单管同步分型的多重套式PCR检测方法相匹配的标准化检测试剂。通过稳定性试验和实验室间验证试验，证明了该检测试剂盒具有良好稳定性和可重复性。
（3）本研究建立了衣原体的实时定量PCR检测方法，灵敏度高，特异性强。最低可以检测200拷贝/μL质粒DNA，可用于衣原体的诊断和流行病学调查。
（4）本文基于ompA基因，建立了衣原体科LAMP检测方法。该方法具有特异性强、灵敏度高及实验条件要求较低的特点，可以在水浴锅中完成，在添加了钙黄绿素后，阳性的LAMP扩增反应呈现明显的黄绿色，使检测结果的非常直观，容易判读。本法适宜于临床上对衣原体病进行现场快速筛查，但阳性样品可用其他方法进行分型确证。
（5）应用本文研发的猪衣原体、流产嗜衣原体及肺炎嗜衣原体的单管同步分型的多重套式PCR检测试剂盒对青海羊场和重庆猪场采集的样品进行检测。首次从猪检测到C.suis和Cpn，首次从我国绵羊和山羊检出C.suis和Cpn，并发现C.suis、Cpn和Ch.abortus对绵羊和山羊的混合感染。
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.
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