猪主要致病链球菌多重PCR诊断方法的建立及流行病学调查应用
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摘要
链球菌种型众多,但致病性差异显著,本研究针对猪的主要致病种和型(C群马链球菌兽疫亚种Streptococcus equi subsp . zooepidemicus和猪链球菌1、2、7、9型Streptococcus suis,SS1、SS2、SS7、SS9),在属、种、型三个水平上建立了一次性诊断链球菌的多重PCR诊断方法;并应用初步建立的多重PCR方法,结合本实验室在流行病学调查时使用的单纯PCR分型方法、血清学分型方法和MLST分型方法,对中国部分地区猪链球菌的流行病学进行调查分析。研究结果如下:
(1)猪主要致病链球菌多重PCR诊断方法的建立:根据GenBank已登录序列,针对链球菌属保守基因(延伸因子EF-TU)、SS种保守基因(谷氨酸脱氢酶,GDH)、马链球菌兽疫亚种保守基因(类M蛋白,M-like)、SS型特异性基因(荚膜多糖,SS1型CPS1I、SS2型CPS2J、SS7型CPS7H和SS9型CPS9H)设计合成7对引物,预计扩增片段大小分别为:197、943、1137、441、291、541、388bp;制备混合模板(SS1、SS2、SS7、SS9和Streptococcus equi subsp . zooepidemicus),建立多重PCR方法。设B群无乳链球菌、变形链球菌、金黄色葡萄球菌、大肠埃希菌和水为对照进行特异性试验。用SS1、SS2、SS7、SS9标准株及Streptococcus equi subsp . zooepidemicus猪源555株进行准确性试验;并对16株链球菌临床分离鉴定菌进行验证性检测。梯度稀释混合模板进行敏感性试验。分别制备混合模板进行5次重复性试验。结果表明,建立的多重PCR方法在退火温度为61℃时可一次性扩出7条符合预期大小、序列正确的基因条带。特异性试验和准确性试验结果均符合预期,无非特异性、交叉反应、假阳性、假阴性条带出现,对16株临床分离鉴定链球菌的验证检测结果和细菌学鉴定及单纯PCR鉴定的符合率为100%。敏感性为0.08ng/μL;5次重复试验结果一致。结果提示,本研究建立的多重PCR方法,是截至目前唯一能够一次性在属、种、型三个水平上同时诊断猪主要致病链球菌的PCR诊断方法,具有高度特异性、准确性、敏感性和稳定性,高效、经济、快速是其优势,适用于猪场链球菌病监控和流行病学快速诊断。
(2)流行病学调查:对2010年6个省份屠宰场的900份扁桃体或淋巴结样品进行细菌分离培养,然后用GDH、CPS1I、CPS2J、CPS7H、CPS9G基因序列PCR鉴定种型,并用血清凝集试验复检,共检出28株猪链球菌(检出率为3.11%);其中3株为SS2,2株SS7,1株SS9,2株SS3,1株SS15,1株SS19,1株SS27,1株SS34;最后用多位点序列分型(Multilocus sequence typing,MLST)系统研究各菌株之间的遗传关系,在13株能够定型的猪链球菌中,3株ST1,2株ST29,2株ST168,ST27、ST28、ST45、ST117、ST163和ST174各1株,两株申报ST新型;部分菌株的7个管家基因(aroA、Cpn60、Dpr、Gki、Muts、RecA、ThrA)中的某些基因的PCR扩增产物呈阴性或不能准确测序,导致无法从MLST数据库中比对出相应的ST型。
Pathogenicity between numerous species and types of Streptococci are strikingly different. Targeting at predominant pig-pathogenic species and types including Streptococcus equi subsp . zooepidemicus and Streptococcus suis type1, 2, 7, 9 (SS1, SS2, SS7, SS9), present study aimed to develop a novel polymerase chain reaction (PCR) method to diagnose Streptococci at genus, species and type levels simultaneously by just a single PCR. And it was applied to carry out epidemiological survey of Streptococcus suis in parts of china by combining with simple PCR, serum agglutination test and MLST typing method. The major results are as follows:
(1)Development of multiple PCR assay for pig-pathogenic of Streptococci. Based on genetic sequences in Genebank, 7 pairs of primers respectively targeting at Streptococcus genus-conservative gene (elongation factor EF-TU), SS and Streptococcus equi subsp . zooepidemicus conservative gene (glutamate dehydrogenase, GDH and M-like protein respectively), and type specific genes (capsular polysacchride, CPS, CPS1I, CPS2J, CPS7H, CPS9H respectively for SS1, SS2, SS7, SS9) were designed with expected length of amplified fragments respectively as 197, 943, 1137, 441, 291, 541, 388bp. Multiple PCR was developed based on template mix(SS1, 2, 7, 9 and Streptococcus equi subsp . zooepidemicus) Specificity of the multiple PCR was determined using S. agalactiae and S.Mutans, Staphylococcus aureus, E.coli and water respectively as control. Accuracy was verified by standard strains of SS1, SS2, SS7, SS9 and Streptococcus equi subsp . zooepidemicus strain 555, and by detecting 16 clinical isolates already identified by traditional bacteriological methods and simple PCR. Sensitivity was confirmed by serial dilution of template mix and stability by preparation of template mix each time. The novel multiple PCR assay designed in this study could clearly produce 7 expected bands with correct sizes and sequences at 61℃as annealing temperature. Specificity and accuracy were confirmed by expected bands when detecting standard SS strains and Streptococcus equi subsp . zooepidemicus strain 555, and even further proven by 100% in agreement with bacteriological and PCR results when detecting 16 Streptococcus isolates. Sensitivity reached 0.08ng/μL and stability were verified by same performance in 5 separate tests. The multiple PCR developed in this study, are the only assay up to date which could diagnose at three levels (genus, species, type) for pig-pathogenic Streptococcus by a single PCR with high specificity, accuracy, sensitivity and stability. The striking advantages of this novel PCR are highly efficient, economic and time-saving, suiting for Streptococcus surveillance in pig farm and fast epidemiological survey.
(2)The epidemiological survey of Streptococcus suis . 900 samples(tonsil or lymph) were collected from 6 provinces of China in 2010. Streptococcus suis were isolated and purified, followed by identification of suspected species and types by PCR based on gene squences of GDH, CPS1I, CPS2J, CPS7H, CPS9G and serum agglutination test. The results showed: of the 28 strains identified, 3 strains are SS2, 2 SS7, 1 SS9, 2 SS3, 1 SS15, 1 SS19, 1 SS27, 1 SS34. Genetic relationships of isolates were studied by multilocus sequence typing (MLST). Analyzed by MLST, The seven housekeeper genes (aroA, Cpn60, Gki, Dpr, Muts, ThrA, RecA) of some strains, the PCR product negative or not accurate sequencing, so can't determine the corresponding ST type. 13 strains can be identified, the results implied that 3 strains are ST1, 2 ST29, 2 ST168, ST27, ST28, ST45, ST117, ST163, ST174 each one strain. Two strains belonged to new ST types, which was not reported in previous publications. The new ST was submitted to MLST database of Streptococcus suis (http://ssuis.mlst.net).
(1)猪主要致病链球菌多重PCR诊断方法的建立:根据GenBank已登录序列,针对链球菌属保守基因(延伸因子EF-TU)、SS种保守基因(谷氨酸脱氢酶,GDH)、马链球菌兽疫亚种保守基因(类M蛋白,M-like)、SS型特异性基因(荚膜多糖,SS1型CPS1I、SS2型CPS2J、SS7型CPS7H和SS9型CPS9H)设计合成7对引物,预计扩增片段大小分别为:197、943、1137、441、291、541、388bp;制备混合模板(SS1、SS2、SS7、SS9和Streptococcus equi subsp . zooepidemicus),建立多重PCR方法。设B群无乳链球菌、变形链球菌、金黄色葡萄球菌、大肠埃希菌和水为对照进行特异性试验。用SS1、SS2、SS7、SS9标准株及Streptococcus equi subsp . zooepidemicus猪源555株进行准确性试验;并对16株链球菌临床分离鉴定菌进行验证性检测。梯度稀释混合模板进行敏感性试验。分别制备混合模板进行5次重复性试验。结果表明,建立的多重PCR方法在退火温度为61℃时可一次性扩出7条符合预期大小、序列正确的基因条带。特异性试验和准确性试验结果均符合预期,无非特异性、交叉反应、假阳性、假阴性条带出现,对16株临床分离鉴定链球菌的验证检测结果和细菌学鉴定及单纯PCR鉴定的符合率为100%。敏感性为0.08ng/μL;5次重复试验结果一致。结果提示,本研究建立的多重PCR方法,是截至目前唯一能够一次性在属、种、型三个水平上同时诊断猪主要致病链球菌的PCR诊断方法,具有高度特异性、准确性、敏感性和稳定性,高效、经济、快速是其优势,适用于猪场链球菌病监控和流行病学快速诊断。
(2)流行病学调查:对2010年6个省份屠宰场的900份扁桃体或淋巴结样品进行细菌分离培养,然后用GDH、CPS1I、CPS2J、CPS7H、CPS9G基因序列PCR鉴定种型,并用血清凝集试验复检,共检出28株猪链球菌(检出率为3.11%);其中3株为SS2,2株SS7,1株SS9,2株SS3,1株SS15,1株SS19,1株SS27,1株SS34;最后用多位点序列分型(Multilocus sequence typing,MLST)系统研究各菌株之间的遗传关系,在13株能够定型的猪链球菌中,3株ST1,2株ST29,2株ST168,ST27、ST28、ST45、ST117、ST163和ST174各1株,两株申报ST新型;部分菌株的7个管家基因(aroA、Cpn60、Dpr、Gki、Muts、RecA、ThrA)中的某些基因的PCR扩增产物呈阴性或不能准确测序,导致无法从MLST数据库中比对出相应的ST型。
Pathogenicity between numerous species and types of Streptococci are strikingly different. Targeting at predominant pig-pathogenic species and types including Streptococcus equi subsp . zooepidemicus and Streptococcus suis type1, 2, 7, 9 (SS1, SS2, SS7, SS9), present study aimed to develop a novel polymerase chain reaction (PCR) method to diagnose Streptococci at genus, species and type levels simultaneously by just a single PCR. And it was applied to carry out epidemiological survey of Streptococcus suis in parts of china by combining with simple PCR, serum agglutination test and MLST typing method. The major results are as follows:
(1)Development of multiple PCR assay for pig-pathogenic of Streptococci. Based on genetic sequences in Genebank, 7 pairs of primers respectively targeting at Streptococcus genus-conservative gene (elongation factor EF-TU), SS and Streptococcus equi subsp . zooepidemicus conservative gene (glutamate dehydrogenase, GDH and M-like protein respectively), and type specific genes (capsular polysacchride, CPS, CPS1I, CPS2J, CPS7H, CPS9H respectively for SS1, SS2, SS7, SS9) were designed with expected length of amplified fragments respectively as 197, 943, 1137, 441, 291, 541, 388bp. Multiple PCR was developed based on template mix(SS1, 2, 7, 9 and Streptococcus equi subsp . zooepidemicus) Specificity of the multiple PCR was determined using S. agalactiae and S.Mutans, Staphylococcus aureus, E.coli and water respectively as control. Accuracy was verified by standard strains of SS1, SS2, SS7, SS9 and Streptococcus equi subsp . zooepidemicus strain 555, and by detecting 16 clinical isolates already identified by traditional bacteriological methods and simple PCR. Sensitivity was confirmed by serial dilution of template mix and stability by preparation of template mix each time. The novel multiple PCR assay designed in this study could clearly produce 7 expected bands with correct sizes and sequences at 61℃as annealing temperature. Specificity and accuracy were confirmed by expected bands when detecting standard SS strains and Streptococcus equi subsp . zooepidemicus strain 555, and even further proven by 100% in agreement with bacteriological and PCR results when detecting 16 Streptococcus isolates. Sensitivity reached 0.08ng/μL and stability were verified by same performance in 5 separate tests. The multiple PCR developed in this study, are the only assay up to date which could diagnose at three levels (genus, species, type) for pig-pathogenic Streptococcus by a single PCR with high specificity, accuracy, sensitivity and stability. The striking advantages of this novel PCR are highly efficient, economic and time-saving, suiting for Streptococcus surveillance in pig farm and fast epidemiological survey.
(2)The epidemiological survey of Streptococcus suis . 900 samples(tonsil or lymph) were collected from 6 provinces of China in 2010. Streptococcus suis were isolated and purified, followed by identification of suspected species and types by PCR based on gene squences of GDH, CPS1I, CPS2J, CPS7H, CPS9G and serum agglutination test. The results showed: of the 28 strains identified, 3 strains are SS2, 2 SS7, 1 SS9, 2 SS3, 1 SS15, 1 SS19, 1 SS27, 1 SS34. Genetic relationships of isolates were studied by multilocus sequence typing (MLST). Analyzed by MLST, The seven housekeeper genes (aroA, Cpn60, Gki, Dpr, Muts, ThrA, RecA) of some strains, the PCR product negative or not accurate sequencing, so can't determine the corresponding ST type. 13 strains can be identified, the results implied that 3 strains are ST1, 2 ST29, 2 ST168, ST27, ST28, ST45, ST117, ST163, ST174 each one strain. Two strains belonged to new ST types, which was not reported in previous publications. The new ST was submitted to MLST database of Streptococcus suis (http://ssuis.mlst.net).
引文
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