结核杆菌Taqman PCR检测方法建立及吉林省鹿结核杆菌MLVA分型研究
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摘要
鹿结核病(Deer TB)是由牛分枝杆菌和人型结核杆菌引起的一种人畜共患传染病。目前全世界人工驯养的鹿大约500万头,鹿结核病的发病率不断上升,给养鹿业带来了巨大的威胁,同时患结核病的鹿也成为人结核病的传染源之一。因此,对鹿结核病的病原检测、基因分型、以及病原耐药性分析对结核病(TB)的防治、公众健康和流行病学研究都非常重要。
本研究为建立结核分枝杆菌快速鉴别检测方法,根据GENEBANK登录的致病性结核分枝杆菌复合群(MTC1)、人型结核杆菌(M1)和牛分枝杆菌(B1)特有基因序列设计合成引物及探针,构建了pEASE-MTC、pEASE-B1、pEASE-M1三个重组质粒,用于建立Taqman PCR标准曲线,实验结果显示三条标准曲线都具有较高相关性,可在103～108Copies/μL浓度内定量检测临床细菌样本,特异性实验显示建立的荧光PCR快速检测方法对标准质控菌株呈阳性反应,对卡介苗(BCG)及其他微生物样品为阴性反应。对结核分枝杆菌检测灵敏度可达单个菌细胞。对45份PPD试验检测为阳性的临床样本进行荧光定量PCR检测时,36份为阳性；而对PPD检测为阴性的50份临床样本进行检测时,7份为阳性。本研究结果表明,所建立的方法能够用于结核杆菌的鉴别检测,可对由BCG接种或环境中分枝杆菌引起的PPD检测假阳性样本进行鉴别,对TB的快速检测和早期诊断具有重要意义。
从吉林省养鹿业富有代表性地区的20个鹿场随机采集血清样本1856份作为研究对象,利用结核杆菌Ag85-East6-mpt70抗原多联表达蛋白作为检测抗原,进行酶联免疫检测,调查吉林省鹿结核病流行情况,结果发现吉林省地区鹿群中结核病存在病例,鹿群结核病感染阳性率为17.83%,其中吉林市为23.22%,是吉林省鹿结核病抗体阳性率最高地区,最低地区是松原地区,阳性率仅为11.33%,各地区间阳性率差异显著(p<0.05),不同性别的鹿群结核病的感染率差异显著,除吉林市外其他地区公鹿的阳性率都高于母鹿,不同年龄鹿群结核病的感染率差异显著,仔鹿感染率最低,为12.47%,与育成鹿和成年鹿差异显著,防疫部门应加强防控,防止疫病扩散。采集Taqman PCR检测阳性鹿的组织样本138份,使用斜面培养基进行细菌分离培养,共培养出60株抗酸染色阳性菌,Taqman PCR方法对分离细菌进行鉴定,结果表明,其中48株为致病性结核杆菌,包括牛分枝杆菌43株,人型结核杆菌5株。43株牛分枝杆菌中有5株分离于结核病ELISA检测阴性的样本,5株人型结核杆菌全部分离于ELISA检测阳性的样本,分离培养结果表明,Taqman PCR检测方法的特异性高于ELISA检测。
评价29个多位点数目可变串联重复序列(MLVA)在吉林省鹿源结核杆菌基因中的多态性,初步建立适合吉林省的MLVA基因分型方法,探讨吉林省地区鹿源结核杆菌的基因型特点。对吉林省鹿群中分离的48株结核分枝杆菌提取基因组DNA。采用PCR方法扩增结核菌基因组中的29个MLVA位点,扩增的产物经毛细管电泳检测后,确定PCR产物片段长度并计算每个菌株不同串联重复位点的拷贝数,评价每个MLVA位点的多态性。利用Hunter-Gaston指数(HGDI)评价不同位点组合对鹿源结核杆菌的分辨能力。MLVA位点多样性(h)分析结果显示：位点QUB3232、QUB11a、QUB18、QUB4156、ETRB、Mtub29和Mtub39无法扩增出稳定的PCR产物,并且在部分菌株中缺失该序列,其余22个位点中,QUB11b位点多态性较好(h≥0.6)。位点ETRA、MIRU4、MIRU23、MIRU31、MIRU39、MIRU40、QUB26、Mtub4、Mtub21为中等多态性(0.6>h>0.3),位点MIRU2、Mtub30和Mtub34多态性较低分别为0.241、0.212和0.037(h≤0.3),其他位点不具有多态性。13个具有多态性的位点与全部22个位点分型能力相同,将48株分离菌分为12个基因型,分型指数H为0.903,多态性超过0.5的6个位点(ETRA、MIRU4、QUB11b. QUB26、Mtub4、Mtub21)将全部菌株分为12个基因型(H=0.903),6个具有多态性的MIRU位点可分为11个基因型(H=0.890), Mtub4、Mtub21、Mtub30和Mtub34位点可将全部菌株分为9个基因型(H=0.857)。实验结果表明,吉林省鹿源结核杆菌与其他国家或地区的结核分枝杆菌在基因分型方面存在差异,应用MLVA分型方法进行鹿结核病分子流行病学研究非常有效,MLVA技术操作简便、重复率高便于普及,可以在鹿结核病流行病学调查研究上发挥重要作用。
为了解鹿源结核分枝杆菌临床分离菌株对利福平(RFP)、异烟肼(INH)和链霉素(SM)的分子耐药机制,本研究采用Almar Blue微板分析法对分离鉴定的48株鹿源结核分枝杆菌进行药敏分析,筛选出1株耐异烟肼菌株,4株耐链霉素菌株,未发现利福平耐药株,PCR扩增耐药株的rpsL基因片段(830bp)和KatG基因片段(626bp)并测序,通过对耐药基因的测序分析,1株异烟肼耐药菌株在315位点发生碱基突变；耐链霉素菌株中有一株人型结核杆菌未检测到基因突变,其他三株在43位的氨基酸密码子发生突变,密码子由AAG变为AGG。耐药检测结果提示：吉林省鹿群中可能存在一定范围、一定程度的耐药结核分枝杆菌的传播,应加强结核病的耐药监测尤其分子水平的监测,有利于早期发现和预防耐药结核分枝杆菌大规模的扩散,对于控制耐药结核病疫情有重要意义。
Deer tuberculosis (TB) is a chronic bacterial disease of animals and humans caused by Mycobacterium bovis, which is caused by M. tuberculosis and M. bovis. Since the world's population of feeding deer is now up to 5 million, there has been an increasing awareness of the potential threat posed by TB to domesticated deer, and Deer TB is a source of infection for human TB. It is important to identify isolates, genotyping and Drug Resistance of the MTC for epidemiologic and public health considerations and M. tuberculosis to optimize treatment.
For establishment a method of rapid specific detection TB, the real-time PCR assays for specific detection of pathogenicity Mycobacterium tuberculosis, Mycobacterium tuberculosis hominis and Mycobacterium bovis were developed. The standard recombinant plasmids pEASE-MTC, pEASE-B1, pEASE-M1 were constructed as reference standards used in absolute quantification assaying. The CT values and ln(x) were highly correlated (MTC R2=0.9998, B1 R2=0.997, M1 R2=0.9991). The dynamic range of measurement is 103-108 Copies/μL. The assays have positive result with Standard quality control strains, the detection result of BCG and microorganisms is negative, and the assays could detect single bacterial cells. Upon detecting 45 clinical samples that were positive in PPD test, the results of Taqman-PCR showed 36 to be positive. However, there were 7 positive samples in Taqman-PCR assay of 50 samples, while they showed negative results in PPD test. The result showed a method of rapid specific identification of TB had been successfully established, and this method might be of great significance in the early diagnosis and rapid detection of TB.
In order to investigate the prevalence of deer Tuberculosis in Jilin province of China, a total of 1856 blood samples from 20 deer farms were examined by ELISA which used Ag85-East6-mpt70 protein as the diagnostic antigen. The result of prevalence survey showed deer tuberculosis exists in Jilin Province at a positive rate of 17.83%, Jilin city had the highest positive rate of 23.22%; the lowest positive rate was 11.33% in the Songyuan region. A significant difference was seen in six geographical areas, different gender and different age groups. The results also showed that prevention and control of the transmission of deer tuberculosis can be enhanced by the efforts of epidemic prevention departments.
60 strains of acid-fast positive bacilli were cultured from 138 Taqman PCR-detected positive tissue samples by 7H9 medium, including 48 strains of MTBC (43 strains of M.bovis and 5 strains of M.tuberculosis),5 strains of M.bovis which were cultured from ELISA negative samples, and 5 strains of M. tuberculosis which were cultured from ELISA positive samples. The culture result showed Taqman PCR is more specificity than ELISA.
Let us assess the diversity of 24 MLVA loci in 48 stains of MTBC isolated from deer, and discuss the characteristic of the Mycobacterum tuberculosis from Deer. The genotypic characteristics of MTBC in Jilin province were investigated using MLVA genotyping method. The MLVA loci were amplified by PCR which used extracted genomic DNA of MTBC isolates as template, the amplification products were analysis by CE. The number of copies of tandem repeats of 24 MLVA loci were confirmed based on the length of PCR amplification products. The diversity of 24 MLVA loci was assessed by the number of repeated sequences. The discriminatory power of different MLVA loci complex was assessed by Hunter-Gaston index(HGDI). The results of diversity analysis showed:QUB11b was highly discriminative(h≥0.6), ETRA, MIRU4, MIRU23, MIRU31, MIRU39, MIRU40, QUB26, Mtub4 and Mtub21 loci were moderately discriminative (0.6>h>0.3), MIRU2, Mtub30 and Mtub34 were poorly discriminative(h≤0.3), while the other loci had no diversity.13 diversity loci showed similar discrimination with all 24 MLVA loci, and 7 different genotypes were identified with a genotypic diversity indice of 0.903. The set of the 6 loci with the most polymorphism (h>0.5; ETRA, MIRU4, QUB11b, QUB26, Mtub4, Mtub21) also had the same power as all 24 MLVA loci. The 6 diversity MIRUs loci resolved the 48 isolates into 11 genotypes, with a genotypicdiversity indice of 0.890.4 diversity Mtubs loci resolved the 48 isolates into 9 genotypes, with a genotypicdiversity indice of 0.857. The result of this study indicated that the MTBC from Deer in JiLin province were different from other regions, and MLVA tecnique is very useful for the epidemiological investigation of Deer tuberculosis, so it will exert important function in the epidemiological investigation of Deer tuberculosis.
A study on Mycobacterium tuberculosis, which clinically isolated the molecular resistance mechanism of RFP, SM and INH, analysed tuberculosis drug susceptibility of 48 strains MTBC by MABA assay. It selected one INH and four SM strains, then amplified and sequenced the rpsL gene fragments(830bp)and KatG gene fragments(626bp) of the 5 strains. The result showed that 1 INH resistant strains eperienced mutation in 315 site of KatG gene(AGC to ACC), while three SM strains had mutation in 43 site of rpsL gene(AAG to AGG) and the other one SM resistant strains did not show any occurrence of mutation. The detection of DR-TB cases indicated that epidemic spreading of DR-TB might exist within a certain scope to some extent in deer farms of Jilin province, so it is necessary to reinforce the surveillance of drug resistance, especially at the molecular level, in order to diagnose the expansion of mycobacterium tuberculosis at an early stage, which is also important to the ability to control the epidemic situation of DR-TB.
本研究为建立结核分枝杆菌快速鉴别检测方法,根据GENEBANK登录的致病性结核分枝杆菌复合群(MTC1)、人型结核杆菌(M1)和牛分枝杆菌(B1)特有基因序列设计合成引物及探针,构建了pEASE-MTC、pEASE-B1、pEASE-M1三个重组质粒,用于建立Taqman PCR标准曲线,实验结果显示三条标准曲线都具有较高相关性,可在103～108Copies/μL浓度内定量检测临床细菌样本,特异性实验显示建立的荧光PCR快速检测方法对标准质控菌株呈阳性反应,对卡介苗(BCG)及其他微生物样品为阴性反应。对结核分枝杆菌检测灵敏度可达单个菌细胞。对45份PPD试验检测为阳性的临床样本进行荧光定量PCR检测时,36份为阳性；而对PPD检测为阴性的50份临床样本进行检测时,7份为阳性。本研究结果表明,所建立的方法能够用于结核杆菌的鉴别检测,可对由BCG接种或环境中分枝杆菌引起的PPD检测假阳性样本进行鉴别,对TB的快速检测和早期诊断具有重要意义。
从吉林省养鹿业富有代表性地区的20个鹿场随机采集血清样本1856份作为研究对象,利用结核杆菌Ag85-East6-mpt70抗原多联表达蛋白作为检测抗原,进行酶联免疫检测,调查吉林省鹿结核病流行情况,结果发现吉林省地区鹿群中结核病存在病例,鹿群结核病感染阳性率为17.83%,其中吉林市为23.22%,是吉林省鹿结核病抗体阳性率最高地区,最低地区是松原地区,阳性率仅为11.33%,各地区间阳性率差异显著(p<0.05),不同性别的鹿群结核病的感染率差异显著,除吉林市外其他地区公鹿的阳性率都高于母鹿,不同年龄鹿群结核病的感染率差异显著,仔鹿感染率最低,为12.47%,与育成鹿和成年鹿差异显著,防疫部门应加强防控,防止疫病扩散。采集Taqman PCR检测阳性鹿的组织样本138份,使用斜面培养基进行细菌分离培养,共培养出60株抗酸染色阳性菌,Taqman PCR方法对分离细菌进行鉴定,结果表明,其中48株为致病性结核杆菌,包括牛分枝杆菌43株,人型结核杆菌5株。43株牛分枝杆菌中有5株分离于结核病ELISA检测阴性的样本,5株人型结核杆菌全部分离于ELISA检测阳性的样本,分离培养结果表明,Taqman PCR检测方法的特异性高于ELISA检测。
评价29个多位点数目可变串联重复序列(MLVA)在吉林省鹿源结核杆菌基因中的多态性,初步建立适合吉林省的MLVA基因分型方法,探讨吉林省地区鹿源结核杆菌的基因型特点。对吉林省鹿群中分离的48株结核分枝杆菌提取基因组DNA。采用PCR方法扩增结核菌基因组中的29个MLVA位点,扩增的产物经毛细管电泳检测后,确定PCR产物片段长度并计算每个菌株不同串联重复位点的拷贝数,评价每个MLVA位点的多态性。利用Hunter-Gaston指数(HGDI)评价不同位点组合对鹿源结核杆菌的分辨能力。MLVA位点多样性(h)分析结果显示：位点QUB3232、QUB11a、QUB18、QUB4156、ETRB、Mtub29和Mtub39无法扩增出稳定的PCR产物,并且在部分菌株中缺失该序列,其余22个位点中,QUB11b位点多态性较好(h≥0.6)。位点ETRA、MIRU4、MIRU23、MIRU31、MIRU39、MIRU40、QUB26、Mtub4、Mtub21为中等多态性(0.6>h>0.3),位点MIRU2、Mtub30和Mtub34多态性较低分别为0.241、0.212和0.037(h≤0.3),其他位点不具有多态性。13个具有多态性的位点与全部22个位点分型能力相同,将48株分离菌分为12个基因型,分型指数H为0.903,多态性超过0.5的6个位点(ETRA、MIRU4、QUB11b. QUB26、Mtub4、Mtub21)将全部菌株分为12个基因型(H=0.903),6个具有多态性的MIRU位点可分为11个基因型(H=0.890), Mtub4、Mtub21、Mtub30和Mtub34位点可将全部菌株分为9个基因型(H=0.857)。实验结果表明,吉林省鹿源结核杆菌与其他国家或地区的结核分枝杆菌在基因分型方面存在差异,应用MLVA分型方法进行鹿结核病分子流行病学研究非常有效,MLVA技术操作简便、重复率高便于普及,可以在鹿结核病流行病学调查研究上发挥重要作用。
为了解鹿源结核分枝杆菌临床分离菌株对利福平(RFP)、异烟肼(INH)和链霉素(SM)的分子耐药机制,本研究采用Almar Blue微板分析法对分离鉴定的48株鹿源结核分枝杆菌进行药敏分析,筛选出1株耐异烟肼菌株,4株耐链霉素菌株,未发现利福平耐药株,PCR扩增耐药株的rpsL基因片段(830bp)和KatG基因片段(626bp)并测序,通过对耐药基因的测序分析,1株异烟肼耐药菌株在315位点发生碱基突变；耐链霉素菌株中有一株人型结核杆菌未检测到基因突变,其他三株在43位的氨基酸密码子发生突变,密码子由AAG变为AGG。耐药检测结果提示：吉林省鹿群中可能存在一定范围、一定程度的耐药结核分枝杆菌的传播,应加强结核病的耐药监测尤其分子水平的监测,有利于早期发现和预防耐药结核分枝杆菌大规模的扩散,对于控制耐药结核病疫情有重要意义。
Deer tuberculosis (TB) is a chronic bacterial disease of animals and humans caused by Mycobacterium bovis, which is caused by M. tuberculosis and M. bovis. Since the world's population of feeding deer is now up to 5 million, there has been an increasing awareness of the potential threat posed by TB to domesticated deer, and Deer TB is a source of infection for human TB. It is important to identify isolates, genotyping and Drug Resistance of the MTC for epidemiologic and public health considerations and M. tuberculosis to optimize treatment.
For establishment a method of rapid specific detection TB, the real-time PCR assays for specific detection of pathogenicity Mycobacterium tuberculosis, Mycobacterium tuberculosis hominis and Mycobacterium bovis were developed. The standard recombinant plasmids pEASE-MTC, pEASE-B1, pEASE-M1 were constructed as reference standards used in absolute quantification assaying. The CT values and ln(x) were highly correlated (MTC R2=0.9998, B1 R2=0.997, M1 R2=0.9991). The dynamic range of measurement is 103-108 Copies/μL. The assays have positive result with Standard quality control strains, the detection result of BCG and microorganisms is negative, and the assays could detect single bacterial cells. Upon detecting 45 clinical samples that were positive in PPD test, the results of Taqman-PCR showed 36 to be positive. However, there were 7 positive samples in Taqman-PCR assay of 50 samples, while they showed negative results in PPD test. The result showed a method of rapid specific identification of TB had been successfully established, and this method might be of great significance in the early diagnosis and rapid detection of TB.
In order to investigate the prevalence of deer Tuberculosis in Jilin province of China, a total of 1856 blood samples from 20 deer farms were examined by ELISA which used Ag85-East6-mpt70 protein as the diagnostic antigen. The result of prevalence survey showed deer tuberculosis exists in Jilin Province at a positive rate of 17.83%, Jilin city had the highest positive rate of 23.22%; the lowest positive rate was 11.33% in the Songyuan region. A significant difference was seen in six geographical areas, different gender and different age groups. The results also showed that prevention and control of the transmission of deer tuberculosis can be enhanced by the efforts of epidemic prevention departments.
60 strains of acid-fast positive bacilli were cultured from 138 Taqman PCR-detected positive tissue samples by 7H9 medium, including 48 strains of MTBC (43 strains of M.bovis and 5 strains of M.tuberculosis),5 strains of M.bovis which were cultured from ELISA negative samples, and 5 strains of M. tuberculosis which were cultured from ELISA positive samples. The culture result showed Taqman PCR is more specificity than ELISA.
Let us assess the diversity of 24 MLVA loci in 48 stains of MTBC isolated from deer, and discuss the characteristic of the Mycobacterum tuberculosis from Deer. The genotypic characteristics of MTBC in Jilin province were investigated using MLVA genotyping method. The MLVA loci were amplified by PCR which used extracted genomic DNA of MTBC isolates as template, the amplification products were analysis by CE. The number of copies of tandem repeats of 24 MLVA loci were confirmed based on the length of PCR amplification products. The diversity of 24 MLVA loci was assessed by the number of repeated sequences. The discriminatory power of different MLVA loci complex was assessed by Hunter-Gaston index(HGDI). The results of diversity analysis showed:QUB11b was highly discriminative(h≥0.6), ETRA, MIRU4, MIRU23, MIRU31, MIRU39, MIRU40, QUB26, Mtub4 and Mtub21 loci were moderately discriminative (0.6>h>0.3), MIRU2, Mtub30 and Mtub34 were poorly discriminative(h≤0.3), while the other loci had no diversity.13 diversity loci showed similar discrimination with all 24 MLVA loci, and 7 different genotypes were identified with a genotypic diversity indice of 0.903. The set of the 6 loci with the most polymorphism (h>0.5; ETRA, MIRU4, QUB11b, QUB26, Mtub4, Mtub21) also had the same power as all 24 MLVA loci. The 6 diversity MIRUs loci resolved the 48 isolates into 11 genotypes, with a genotypicdiversity indice of 0.890.4 diversity Mtubs loci resolved the 48 isolates into 9 genotypes, with a genotypicdiversity indice of 0.857. The result of this study indicated that the MTBC from Deer in JiLin province were different from other regions, and MLVA tecnique is very useful for the epidemiological investigation of Deer tuberculosis, so it will exert important function in the epidemiological investigation of Deer tuberculosis.
A study on Mycobacterium tuberculosis, which clinically isolated the molecular resistance mechanism of RFP, SM and INH, analysed tuberculosis drug susceptibility of 48 strains MTBC by MABA assay. It selected one INH and four SM strains, then amplified and sequenced the rpsL gene fragments(830bp)and KatG gene fragments(626bp) of the 5 strains. The result showed that 1 INH resistant strains eperienced mutation in 315 site of KatG gene(AGC to ACC), while three SM strains had mutation in 43 site of rpsL gene(AAG to AGG) and the other one SM resistant strains did not show any occurrence of mutation. The detection of DR-TB cases indicated that epidemic spreading of DR-TB might exist within a certain scope to some extent in deer farms of Jilin province, so it is necessary to reinforce the surveillance of drug resistance, especially at the molecular level, in order to diagnose the expansion of mycobacterium tuberculosis at an early stage, which is also important to the ability to control the epidemic situation of DR-TB.
引文
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