猕猴源肺炎链球菌分离和鉴定及FQ-PCR建立和在阐明其侵染机制研究中的应用
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摘要
1猕猴源肺炎链球菌菌株的分离和鉴定
某猕猴繁殖与饲养场发生猕猴突然死亡的疾病,经临床观察、尸检,细菌的分离与鉴定及对小鼠的致病性试验,初步诊断为肺炎链球菌感染。
2肺炎链球菌种特异性PCR和FQ-PCR方法的建立
肺炎链球菌(Streptococcus pneurnoniae, SP)感染是一种重要的人畜共患病,本文根据GenBank登录的SP种特异性DNA序列,设计合成了一对能特异性扩增818 bp DNA片段的引物首次建立了SP种特异性的聚合酶链式反应(PCR)诊断方法；与此同时,设计合成了一对能特异性扩增96bp DNA片段的引物,首次建立了SP种特异性的实时荧光定量聚合酶链式反应(FQ-PCR)诊断方法。此两种方法只能特异性扩增SP,而对草绿色链球菌ATCC49456、口腔链球菌ATCC35037、咽峡炎链球菌ATCC33397、金黄色葡萄球菌、铜绿假单胞菌、大肠埃希菌、嗜肺军团菌、肺炎克雷伯杆菌、粪肠球菌、屎肠球菌等常见人兽共患呼吸道致病菌的扩增结果均为阴性；FQ-PCR诊断方法建立的标准曲线为Y=-3.357X+48.485,相关系数为1.000,PCR循环效率为98.5%；检测SP标准DNA模板的灵敏度为6copies/μL,检测SP细菌数的敏感性为8CFU/μL,重复性差异系数为1.8%。分别应用该技术和传统细菌分离法检测人工感染死亡小鼠的心、肝、脑和肺脏,结果显示两种方法对心、肝、肺脏的阳性检测结果符合率为100%,而FQ-PCR对脑的阳性检测率极显著(P<0.01)高于细菌分离。研究结果表明,该方法快速、灵敏、特异、重复性好且能实时定量检测,可用于SP分离鉴定、SP感染疑似病例检测、SP体内动态分布规律研究及其分子流行病学调查。
3在肺炎链球菌实验感染小鼠侵染机制研究中的应用
应用建立的FQ-PCR方法对经腹腔接种和滴鼻途经人工感染SP小鼠组织器官的检测结果表明：
经腹腔接种该菌后最早可在2h内从血液中检测到,4h能在心、肺、肝和脾中检测到；8h后可在除十二指肠、空肠、回肠、盲肠、直肠、胸腺、胰、鼻、咽喉、气管和脑外各组织器官检测到。24-36h之间各组织器官均能检测到SP且含菌量达到最高,统计显示,心、肺、肝脏、肾脏、脾脏、胰、脑和血的阳性拷贝数高于其它部位达到3-4个数量级,差异极显著,通过此途径接种的小鼠在36h后全部死亡。
滴鼻组中感染小鼠SP的DNA拷贝数显著低于前一种感染途径。2h在肺中最早检测到,4h后能在血液中被检测到,12h可在除胰、十二指肠、空肠、回肠、盲肠、直肠、脑外的所有组织器官中被检测到,但拷贝数高于腹腔注射组达1-2个数量级。24-36h后全部被检组织器官呈阳性反应且达到最高峰；同样地,心、血、肺脏、肝脏、脾脏、’肾脏的阳性拷贝数明显高于其它部位。直到48h后含菌量开始下降2-3个数量级或不能被检测到阳性结果。然而该菌能在脾、鼻、咽喉、气管和肺中持续到9d仍能被检测到,接种第9天时以肺的含菌量最高。
SP经两种途径感染后的共同特点是：呼吸系统中以鼻和肺脏的含菌量最高,且能持续存在9d而不被机体清除；主要内脏器官中以心、脾的含菌量较高,且能在脾中持续存在9d而不被机体所清除；该菌感染宿主后能迅速作用于免疫器官,其中以脾的含菌量显著高于其它器官；脑出现阳性结果的时间较晚,但阳性拷贝数到后期逐渐升高。本次的研究结果能对SP的感染侵入机制提供了进一步的认识。
1 Isolation and identification of bacteria from Rhesus monkey
The Streptococcus pneumoniae disease occurred among rhesus monkeys(Macaca mulatta) suddenly and clusters of sudden deaths have been observed in a rhesus monkeys reproduce and breed farm in Sichuan province of China. According to the results of the clinical observation, necropsies, isolation and identification of bacteria and pathogenicity to mice, we conclude that the agent is Streptococcus pneumoniae.
2 The development of species specific PCR and FQ-PCR for Streptococcus pneumoniae
Streptococcus pneurnoniae (SP) is one of the important zoonosis. Ply, encoding the cytolysin pneumolysin on the species specific DNA sequence of SP from GenBank, the specific primers which showed 818 bp fragment for PCR,96bp fragment for FQ-PCR, and the species specific PCR method and FQ-PCR method for SP detection was developed initially. Applied this assay to Streptococcus including Streptococcus anginosus ATCC 33397,Streptococcus mitis ATCC 49456, Streptococcus oralis ATCC 35037, and several common pathogenic bacterias of nasopharynx such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis,Enterococcus faecium indicated that it was highly specific to SP, there was no amplification with no SP genera. Also, a series of sensitivity experiments were performed and proved that the detection limit of this method was 6 copies/μL for genomic DNA,8CFU/μL for bacterial number and the standard deviation was 1.8%.The correlation coefficient for the associated standard curve was 1.000 and PCR efficiency was 98.5%, the formula was Y=-3.357X+48.485.Two artificial infected cases which were infected with SP were chosen to identify the accuracy and sensitivity of the PCR and FQ-PCR method after death.Results showed that the detection of heart,liver, brain and lung were identical between bacteria isolating and FQ-PCR. Used this assay to detect the brain indicated that the positive rates were significant difference(P<0.01),the FQ-PCR method was more sensitive. Thus, this FQ-PCR assay provides a more rapid and accurate method for identification of SP than traditional isolation methods. It will help to the isolation and identification of SP，detecting of SP infection suspected cases and investigation of molecular epidemiology.
3 Study on the invasion mechanism of Streptococcus pneumoniae with artificial infected mice
We used this FQ-PCR assay to detect genomic DNA of SP for the artificially infected mice and the results showed that:
After inoculated by intraperitoneally,the blood was positive at 2 h post inoculation, and the heart, lung, liver, spleen were positive at 4h. All the samples were positive at 8h, except duodenum, jejunum, ileum, cecum, rectum,thymus, pancreas, nose, throat, trachea,and brain. The copy number of SP DNA in each tissue reached a peak at 24-36 h post inoculation, with the heart, lung, liver, kidney, spleen, pancreas, brain and blood containing high concentrations of SP, with copies of SP being～1000-10000 times more than those in other regions. However, all the mice were dead at 36h.
After inoculated by nose dripping, the copies of SP was lower than the former route. The lung was positive at 2 h post inoculation, and the blood was positive after 4h. All the samples were positive at 12h,except pancreas, duodenum, jejunum, ileum, cecum,rectum and brain,with copies of SP being-10-100 times more than former infected rout. The copy number of SP DNA in each tissue reached a peak at 24-36 h post inoculation, with the heart, lung, liver, spleen, kidney containing high concentrations of SP than those in other regions.It was still present up to 9 d post inoculation in the spleen, nose, throat, trachea and lung, without causing apparent symptoms.
The common of the two infected routes were as followed, nose and lung had the highest copies number in the respiratory system, and it was still present up to 9 d; With the heart and spleen containing the highest concentrations of SP in the interal organs over the 9d period. Spleen was the primary site for invasion in immune organs of normal mice after challenge.The final organ to show a positive result was the brain at 24 h post inoculation, with low concentrations. This study will help to understand the mechanisms of action of SP infection in vivo.
某猕猴繁殖与饲养场发生猕猴突然死亡的疾病,经临床观察、尸检,细菌的分离与鉴定及对小鼠的致病性试验,初步诊断为肺炎链球菌感染。
2肺炎链球菌种特异性PCR和FQ-PCR方法的建立
肺炎链球菌(Streptococcus pneurnoniae, SP)感染是一种重要的人畜共患病,本文根据GenBank登录的SP种特异性DNA序列,设计合成了一对能特异性扩增818 bp DNA片段的引物首次建立了SP种特异性的聚合酶链式反应(PCR)诊断方法；与此同时,设计合成了一对能特异性扩增96bp DNA片段的引物,首次建立了SP种特异性的实时荧光定量聚合酶链式反应(FQ-PCR)诊断方法。此两种方法只能特异性扩增SP,而对草绿色链球菌ATCC49456、口腔链球菌ATCC35037、咽峡炎链球菌ATCC33397、金黄色葡萄球菌、铜绿假单胞菌、大肠埃希菌、嗜肺军团菌、肺炎克雷伯杆菌、粪肠球菌、屎肠球菌等常见人兽共患呼吸道致病菌的扩增结果均为阴性；FQ-PCR诊断方法建立的标准曲线为Y=-3.357X+48.485,相关系数为1.000,PCR循环效率为98.5%；检测SP标准DNA模板的灵敏度为6copies/μL,检测SP细菌数的敏感性为8CFU/μL,重复性差异系数为1.8%。分别应用该技术和传统细菌分离法检测人工感染死亡小鼠的心、肝、脑和肺脏,结果显示两种方法对心、肝、肺脏的阳性检测结果符合率为100%,而FQ-PCR对脑的阳性检测率极显著(P<0.01)高于细菌分离。研究结果表明,该方法快速、灵敏、特异、重复性好且能实时定量检测,可用于SP分离鉴定、SP感染疑似病例检测、SP体内动态分布规律研究及其分子流行病学调查。
3在肺炎链球菌实验感染小鼠侵染机制研究中的应用
应用建立的FQ-PCR方法对经腹腔接种和滴鼻途经人工感染SP小鼠组织器官的检测结果表明：
经腹腔接种该菌后最早可在2h内从血液中检测到,4h能在心、肺、肝和脾中检测到；8h后可在除十二指肠、空肠、回肠、盲肠、直肠、胸腺、胰、鼻、咽喉、气管和脑外各组织器官检测到。24-36h之间各组织器官均能检测到SP且含菌量达到最高,统计显示,心、肺、肝脏、肾脏、脾脏、胰、脑和血的阳性拷贝数高于其它部位达到3-4个数量级,差异极显著,通过此途径接种的小鼠在36h后全部死亡。
滴鼻组中感染小鼠SP的DNA拷贝数显著低于前一种感染途径。2h在肺中最早检测到,4h后能在血液中被检测到,12h可在除胰、十二指肠、空肠、回肠、盲肠、直肠、脑外的所有组织器官中被检测到,但拷贝数高于腹腔注射组达1-2个数量级。24-36h后全部被检组织器官呈阳性反应且达到最高峰；同样地,心、血、肺脏、肝脏、脾脏、’肾脏的阳性拷贝数明显高于其它部位。直到48h后含菌量开始下降2-3个数量级或不能被检测到阳性结果。然而该菌能在脾、鼻、咽喉、气管和肺中持续到9d仍能被检测到,接种第9天时以肺的含菌量最高。
SP经两种途径感染后的共同特点是：呼吸系统中以鼻和肺脏的含菌量最高,且能持续存在9d而不被机体清除；主要内脏器官中以心、脾的含菌量较高,且能在脾中持续存在9d而不被机体所清除；该菌感染宿主后能迅速作用于免疫器官,其中以脾的含菌量显著高于其它器官；脑出现阳性结果的时间较晚,但阳性拷贝数到后期逐渐升高。本次的研究结果能对SP的感染侵入机制提供了进一步的认识。
1 Isolation and identification of bacteria from Rhesus monkey
The Streptococcus pneumoniae disease occurred among rhesus monkeys(Macaca mulatta) suddenly and clusters of sudden deaths have been observed in a rhesus monkeys reproduce and breed farm in Sichuan province of China. According to the results of the clinical observation, necropsies, isolation and identification of bacteria and pathogenicity to mice, we conclude that the agent is Streptococcus pneumoniae.
2 The development of species specific PCR and FQ-PCR for Streptococcus pneumoniae
Streptococcus pneurnoniae (SP) is one of the important zoonosis. Ply, encoding the cytolysin pneumolysin on the species specific DNA sequence of SP from GenBank, the specific primers which showed 818 bp fragment for PCR,96bp fragment for FQ-PCR, and the species specific PCR method and FQ-PCR method for SP detection was developed initially. Applied this assay to Streptococcus including Streptococcus anginosus ATCC 33397,Streptococcus mitis ATCC 49456, Streptococcus oralis ATCC 35037, and several common pathogenic bacterias of nasopharynx such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis,Enterococcus faecium indicated that it was highly specific to SP, there was no amplification with no SP genera. Also, a series of sensitivity experiments were performed and proved that the detection limit of this method was 6 copies/μL for genomic DNA,8CFU/μL for bacterial number and the standard deviation was 1.8%.The correlation coefficient for the associated standard curve was 1.000 and PCR efficiency was 98.5%, the formula was Y=-3.357X+48.485.Two artificial infected cases which were infected with SP were chosen to identify the accuracy and sensitivity of the PCR and FQ-PCR method after death.Results showed that the detection of heart,liver, brain and lung were identical between bacteria isolating and FQ-PCR. Used this assay to detect the brain indicated that the positive rates were significant difference(P<0.01),the FQ-PCR method was more sensitive. Thus, this FQ-PCR assay provides a more rapid and accurate method for identification of SP than traditional isolation methods. It will help to the isolation and identification of SP，detecting of SP infection suspected cases and investigation of molecular epidemiology.
3 Study on the invasion mechanism of Streptococcus pneumoniae with artificial infected mice
We used this FQ-PCR assay to detect genomic DNA of SP for the artificially infected mice and the results showed that:
After inoculated by intraperitoneally,the blood was positive at 2 h post inoculation, and the heart, lung, liver, spleen were positive at 4h. All the samples were positive at 8h, except duodenum, jejunum, ileum, cecum, rectum,thymus, pancreas, nose, throat, trachea,and brain. The copy number of SP DNA in each tissue reached a peak at 24-36 h post inoculation, with the heart, lung, liver, kidney, spleen, pancreas, brain and blood containing high concentrations of SP, with copies of SP being～1000-10000 times more than those in other regions. However, all the mice were dead at 36h.
After inoculated by nose dripping, the copies of SP was lower than the former route. The lung was positive at 2 h post inoculation, and the blood was positive after 4h. All the samples were positive at 12h,except pancreas, duodenum, jejunum, ileum, cecum,rectum and brain,with copies of SP being-10-100 times more than former infected rout. The copy number of SP DNA in each tissue reached a peak at 24-36 h post inoculation, with the heart, lung, liver, spleen, kidney containing high concentrations of SP than those in other regions.It was still present up to 9 d post inoculation in the spleen, nose, throat, trachea and lung, without causing apparent symptoms.
The common of the two infected routes were as followed, nose and lung had the highest copies number in the respiratory system, and it was still present up to 9 d; With the heart and spleen containing the highest concentrations of SP in the interal organs over the 9d period. Spleen was the primary site for invasion in immune organs of normal mice after challenge.The final organ to show a positive result was the brain at 24 h post inoculation, with low concentrations. This study will help to understand the mechanisms of action of SP infection in vivo.
引文
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