本文選題：高分辨率熔解曲線技術(shù)　切入點(diǎn)：院內(nèi)感染細(xì)菌　出處：《蘭州大學(xué)》2017年碩士論文

【摘要】：目的建立院內(nèi)感染常見9種細(xì)菌大腸埃希菌、銅綠假單胞菌、陰溝腸桿菌、鮑曼不動(dòng)桿菌、肺炎克雷伯菌、表皮葡萄球菌、金黃色葡萄球菌、糞腸球菌和屎腸球菌16S rDNA基因的聚合酶鏈?zhǔn)椒磻?yīng)-高分辨率熔解曲線(PCR-HRM)的分子分型方法,并收集包含在上述9種菌內(nèi)的25株院內(nèi)感染細(xì)菌臨床分離株進(jìn)行PCR-HRM方法臨床驗(yàn)證。方法從文獻(xiàn)中檢索16S rDNA基因V1、V3、V6、V9高變區(qū)引物,收集9種院感細(xì)菌的標(biāo)準(zhǔn)株,使用上述引物擴(kuò)增這9種菌對引物進(jìn)行驗(yàn)證;擴(kuò)增V3-V6區(qū)測序,測序結(jié)果上傳Genbank進(jìn)行同源性比對和分析,確定本實(shí)驗(yàn)所用的標(biāo)準(zhǔn)菌型別與Genbank中所報(bào)道的細(xì)菌種屬一致;對9種院感細(xì)菌標(biāo)準(zhǔn)株的16S rDNA基因V1、V3、V6、V9可變區(qū)進(jìn)行PCR-HRM,并根據(jù)以上各可變區(qū)的HRM熔解曲線形狀和Tm值差異建立亞組分析決定樹方法,分步對院內(nèi)感染常見的9種細(xì)菌鑒別;收集已確定為院內(nèi)感染細(xì)菌并包含在上述9種菌的臨床分離株25株,使用本實(shí)驗(yàn)建立的亞組分析決定樹方法進(jìn)行盲法鑒定,鑒定結(jié)果與微生物表型鑒定結(jié)果進(jìn)行比較。結(jié)果(1)可使用V3、V6、V9區(qū)引物擴(kuò)增9種菌的目的產(chǎn)物,V1區(qū)引物只能用于擴(kuò)增4種革蘭陽性菌(表皮葡萄球菌、金黃色葡萄球菌、糞腸球菌和屎腸球菌);本實(shí)驗(yàn)所用的菌株與Genbank中其相應(yīng)模式菌的相似性為99%-100%,可認(rèn)為所選用菌株的種屬無誤。(2)根據(jù)9種院感細(xì)菌標(biāo)準(zhǔn)株的16S rDNA基因V1、V3、V6、V9可變區(qū)的PCR-HRM結(jié)果,建立了亞組分析決定樹方法:(1)在V1區(qū)鑒別革蘭氏陽性菌和陰性菌:4種革蘭氏陽性菌在V1區(qū)有目的產(chǎn)物,5種革蘭氏陰性菌無目的產(chǎn)物;(2)4種革蘭陽性菌鑒別:在V9區(qū)鑒別葡萄球菌(表皮葡萄球菌和金黃色葡萄球菌)和腸球菌(糞腸球菌和屎腸球菌):葡萄球菌和腸球菌在V9區(qū)按Tm值不同分成兩群(葡萄球菌平均Tm=88.20℃;腸球菌平均Tm=89.75℃);在V3區(qū)鑒別兩種葡萄球菌:兩種葡萄球菌在V3區(qū)熔解曲線形狀不同;在V1區(qū)鑒別兩種腸球菌:兩種腸球菌在V1區(qū)內(nèi)Tm值相差較大(屎腸球菌Tm=85.74℃;糞腸球菌Tm=86.94℃);(3)5種革蘭氏陰性菌鑒別:5種菌在V3區(qū)按照熔解曲線形狀和Tm值的差異分成3群,包括1群(肺炎克雷伯菌和鮑曼不動(dòng)桿菌),2群(陰溝腸桿菌和大腸埃希菌),3群(銅綠假單胞菌);3群的熔解曲線為雙峰,且Tm值較高,可在V3區(qū)直接區(qū)分;在V6區(qū)鑒別1群細(xì)菌:兩種菌在V6區(qū)Tm值差異明顯(肺炎克雷伯菌Tm=86.20℃;鮑曼不動(dòng)桿菌Tm=84.84℃);混熔鑒別2群細(xì)菌:2群細(xì)菌在4個(gè)可變區(qū)的Tm值和曲線形狀差異均不明顯,因此設(shè)置大腸埃希菌為參考菌株,選擇V6區(qū)為擴(kuò)增區(qū),與陰溝腸桿菌V6區(qū)擴(kuò)增產(chǎn)物混熔,結(jié)果顯示為雙峰。(3)25株臨床分離株的驗(yàn)證結(jié)果顯示,22株細(xì)菌的HRM鑒定結(jié)果與微生物表型鑒定結(jié)果一致,1株鑒定結(jié)果不一致(HRM鑒定為金黃色葡萄球菌,微生物表型鑒定結(jié)果為表皮葡萄球菌),2株無法使用亞組分析決定樹分組。結(jié)論利用PCR-HRM技術(shù)建立了院內(nèi)感染常見9種細(xì)菌鑒定的亞組分析決定樹方法;使用亞組分析決定樹方法對9種院感細(xì)菌臨床標(biāo)本鑒定的準(zhǔn)確率為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紓鍒嗘垚3緹，

本文編號：1728110