院内感染细菌16S rDNA基因分型高分辨熔解曲线方法的建立

发布时间：2018-04-09 20:54

  本文选题：高分辨率熔解曲线技术　切入点：院内感染细菌　出处：《兰州大学》2017年硕士论文

【摘要】：目的建立院内感染常见9种细菌大肠埃希菌、铜绿假单胞菌、阴沟肠杆菌、鲍曼不动杆菌、肺炎克雷伯菌、表皮葡萄球菌、金黄色葡萄球菌、粪肠球菌和屎肠球菌16S rDNA基因的聚合酶链式反应-高分辨率熔解曲线(PCR-HRM)的分子分型方法,并收集包含在上述9种菌内的25株院内感染细菌临床分离株进行PCR-HRM方法临床验证。方法从文献中检索16S rDNA基因V1、V3、V6、V9高变区引物,收集9种院感细菌的标准株,使用上述引物扩增这9种菌对引物进行验证;扩增V3-V6区测序,测序结果上传Genbank进行同源性比对和分析,确定本实验所用的标准菌型别与Genbank中所报道的细菌种属一致;对9种院感细菌标准株的16S rDNA基因V1、V3、V6、V9可变区进行PCR-HRM,并根据以上各可变区的HRM熔解曲线形状和Tm值差异建立亚组分析决定树方法,分步对院内感染常见的9种细菌鉴别;收集已确定为院内感染细菌并包含在上述9种菌的临床分离株25株,使用本实验建立的亚组分析决定树方法进行盲法鉴定,鉴定结果与微生物表型鉴定结果进行比较。结果(1)可使用V3、V6、V9区引物扩增9种菌的目的产物,V1区引物只能用于扩增4种革兰阳性菌(表皮葡萄球菌、金黄色葡萄球菌、粪肠球菌和屎肠球菌);本实验所用的菌株与Genbank中其相应模式菌的相似性为99%-100%,可认为所选用菌株的种属无误。(2)根据9种院感细菌标准株的16S rDNA基因V1、V3、V6、V9可变区的PCR-HRM结果,建立了亚组分析决定树方法:(1)在V1区鉴别革兰氏阳性菌和阴性菌:4种革兰氏阳性菌在V1区有目的产物,5种革兰氏阴性菌无目的产物;(2)4种革兰阳性菌鉴别:在V9区鉴别葡萄球菌(表皮葡萄球菌和金黄色葡萄球菌)和肠球菌(粪肠球菌和屎肠球菌):葡萄球菌和肠球菌在V9区按Tm值不同分成两群(葡萄球菌平均Tm=88.20℃;肠球菌平均Tm=89.75℃);在V3区鉴别两种葡萄球菌:两种葡萄球菌在V3区熔解曲线形状不同;在V1区鉴别两种肠球菌:两种肠球菌在V1区内Tm值相差较大(屎肠球菌Tm=85.74℃;粪肠球菌Tm=86.94℃);(3)5种革兰氏阴性菌鉴别:5种菌在V3区按照熔解曲线形状和Tm值的差异分成3群,包括1群(肺炎克雷伯菌和鲍曼不动杆菌),2群(阴沟肠杆菌和大肠埃希菌),3群(铜绿假单胞菌);3群的熔解曲线为双峰,且Tm值较高,可在V3区直接区分;在V6区鉴别1群细菌:两种菌在V6区Tm值差异明显(肺炎克雷伯菌Tm=86.20℃;鲍曼不动杆菌Tm=84.84℃);混熔鉴别2群细菌:2群细菌在4个可变区的Tm值和曲线形状差异均不明显,因此设置大肠埃希菌为参考菌株,选择V6区为扩增区,与阴沟肠杆菌V6区扩增产物混熔,结果显示为双峰。(3)25株临床分离株的验证结果显示,22株细菌的HRM鉴定结果与微生物表型鉴定结果一致,1株鉴定结果不一致(HRM鉴定为金黄色葡萄球菌,微生物表型鉴定结果为表皮葡萄球菌),2株无法使用亚组分析决定树分组。结论利用PCR-HRM技术建立了院内感染常见9种细菌鉴定的亚组分析决定树方法;使用亚组分析决定树方法对9种院感细菌临床标本鉴定的准确率为88%(22/25)。
[Abstract]:Objective to establish 9 kinds of common bacteria Escherichia coli infection, Pseudomonas aeruginosa, Enterobacter cloacae, Bauman Acinetobacter, Klebsiella pneumoniae, Staphylococcus epidermidis, Staphylococcus aureus, polymerase chain reaction of Enterococcus faecalis and Enterococcus faecium 16S gene rDNA high resolution melting (PCR-HRM) molecule typing method, and collected included in the above-mentioned 9 kinds of bacteria in 25 strains of bacteria of nosocomial infection in clinical isolates of PCR-HRM. Methods clinical verification method to retrieve the 16S rDNA gene V1 from the literature V3, V6, V9 hypervariable region primers, collect 9 kinds of nosocomial bacterial strains, amplification of the 9 kinds of bacteria to verify the primer of the primer amplification; V3-V6 sequencing, sequencing results upload Genbank homology comparison and analysis, to determine the bacterial species reported consistent with Genbank type standard strains used in this experiment in 9 kinds of bacteria; nosocomial strains 16S RDNA gene V1, V3, V6, PCR-HRM and V9 variable region, based on the above variable region of the HRM melting curve shape and Tm values were established a subgroup analysis decision tree method, 9 kinds of common bacteria identification step of nosocomial infection; the collection has been identified as bacterial infection in the hospital and included in the 9 from the 25 clinical isolates, using the established subgroup analysis decision tree method for blind identification, identification results of microbial and phenotypic identification results were compared. Results (1) the use of V3, V6, V9 product amplified region 9 strains, V1 primers amplified 4 can only be used for leather gram positive bacteria (Staphylococcus aureus, Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium); similarity of the corresponding model and Genbank strains used in this experiment in 99%-100%, that selected strains are correct. (2) according to the 9 kinds of hospital infection bacteria strain 16S rDNA V3, V6, V1 gene, V9 variable region of PCR-HRM results, a subgroup analysis of decision tree method: (1) in the V1 region of identification of Gram-positive bacteria and Gram-negative bacteria, 4 gram positive bacteria to product in the V1 region, 5 gram negative bacteria to product; (2) 4 kinds of leather gram positive bacteria in V9 identification: identification of Staphylococcus (Staphylococcus epidermidis and Staphylococcus aureus) and enterococci (Enterococcus faecalis and Enterococcus faecium): Staphylococcus aureus and Enterococcus by Tm value in V9 district were divided into two groups (Staphylococcus aureus Enterococcus Tm=89.75 average average Tm=88.20 C; c); in the V3 area to identify two two kinds of Staphylococcus aureus: shape in the V3 region in the V1 region of different melting curve; identification of two Enterococcus: two species of Enterococcus in V1 region Tm value difference (Tm=85.74 Enterococcus faecalis Tm=86.94 C; c); (3) 5 gram negative bacteria identification: 5 kinds of bacteria in the V3 area in accordance with the melting the shape of the curve 鍜孴m鍊肩殑宸�寮傚垎鎴�3缇，

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