鹦鹉热嗜性衣原体特异性TaqMan-MGB探针荧光定量PCR检测方法的建立与应用

目的建立一种特异、灵敏、重复性好的鹦鹉热嗜性衣原体的TaqMan-MGB荧光定量PCR检测方法。方法利用鹦鹉热嗜性衣原体MOMP基因的特异保守序列设计引物和MGB探针,通过优化,获得最佳反应体系与反应条件,同时进行特异性、重复性、灵敏性评价与Spike-test实验进行临床应用性评价,利用该检测方法对禽鸟类粪便样品进行检测,并与常规PCR检测方法进行比较。结果该方法在0.01pg~100pg范围内线性相关系数为0.999,扩增效率为97.7%;对鹦鹉热嗜性衣原体菌株检测结果均呈现阳性,而非鹦鹉热嗜性衣原体菌株及其它衣原体菌株为阴性;重复性试验中,变异系数为0.317%~0.563%;检测灵敏性为0.01pg;Spike-test试验中最低检出量为25个EB;该方法对禽鸟类粪便样品的检出率为14.3%(40/282),高于常规PCR检测法的7.4%(21/282)。结论本文所建立的Taqman-MGB荧光定量PCR检测法是一种灵敏、高效、稳定,可在嗜性衣原体属中准确检测鹦鹉热嗜性衣原体的方法,该方法的建立更有意于今后对禽鸟类实施鹦鹉热的临床诊断与分子流行病学研究。     

沙眼衣原体荧光定量PCR检测方法的建立

目的建立沙眼衣原体实时荧光定量PCR检测技术,以期用于沙眼衣原体的感染检测。方法针对沙眼衣原体ompA基因序列保守区域设计引物和TaqMan探针,构建标准质粒并制作标准曲线,建立沙眼衣原体实时荧光定量检测方法,通过对人型支原体等的检测评价方法的特异性。结果建立的沙眼衣原体荧光定量PCR体系能特异性检测沙眼衣原体,与人型支原体、解脲脲原体、淋球菌、大肠埃希菌EDL933、金黄色葡萄球菌无交叉反应;标准曲线在3.9×109～3.9×103 copies/μl之间线性关系良好(r2=0.99);方法的灵敏度高,检测最低拷贝数为3.9copies/μl;组内及组间变异系数均5%。结论建立的检测沙眼衣原体实时定量PCR方法特异、灵敏,可用于沙眼衣原体感染的早期筛查和临床快速诊断。     

荧光定量PCR检测嗜吞噬细胞无形体

目的采用新型TaqMan-MGB探针建立检测嗜吞噬细胞无形体的实时荧光定量PCR(quantitativereal-timePCR)方法。方法依据gltA基因序列设计嗜吞噬细胞无形体特异引物和探针,以克隆的嗜吞噬细胞无形体gltA基因片段作DNA模板,在荧光定量PCR检测仪(ABI7900HT)上建立实时荧光定量检测方法。结果建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.996);与套式PCR相比较,荧光定量PCR检测的灵敏度是其100倍。用荧光定量PCR检测其它相关立克次体和细菌DNA样本,检出结果几乎为0;对荧光定量PCR检测重复性进行分析,变异系数(CV)批内和批间误差在0~2.1%之间,证明该荧光定量PCR具有种特异性和良好的重复性。用荧光定量PCR检测体疑染嗜吞噬细胞无形体的10份蜱和30份小鼠脾脏标本,结果与套式PCR检测结果有密切相关性,但是定量PCR检测敏感性和准确率均高于套式PCR。结论本研究建立的检测嗜吞噬细胞无形体的实时荧光定量PCR具有很高的特异性和敏感性,特别适合检出样本中微量嗜吞噬细胞无形体。     

鹦鹉热嗜衣原体两对荧光定量PCR引物的应用与比较

针对鹦鹉热嗜衣原体(Cps)的主要外膜蛋白(MOMP)基因和多形态膜蛋白(PMP)基因分别设计引物,建立荧光定量PCR方法,比较两对引物的灵敏度和特异性。试验结果表明,两对引物均可用于Cps检测,在样本中靶标浓度高时,PMP引物的检测灵敏度优于MOMP引物;但前者的扩增效率低于后者,后者更适合用于Cps的实时定量PCR检测。相对定量研究表明国内不同Cps流行株的PMP基因在基因组上的拷贝数可能存在较大差异。     

检测贝氏柯克斯体的实时荧光定量PCR

目的采用新型TaqMan-MGB探针建立检测贝氏柯克斯体的实时荧光定量PCR(real-timequantitativePCR)方法。方法根据贝氏柯克斯体特异的23SrRNA插入基因序列设计引物和探针,以克隆的23SrRNA插入基因片段作DNA模板,在荧光定量PCR检测仪(ABI7900型)上建立实时荧光定量检测方法。结果建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.997);与套式PCR相比较,荧光定量PCR检测敏感性是其100倍。用荧光定量PCR检测其它相关立克次体,检出结果均为0。用荧光定量PCR检测贝氏柯克斯体感染的小鼠脾脏标本,脾脏中贝氏柯克斯体的感染量与感染过程具有相关性。结论本研究建立的检测贝氏柯克斯体的实时荧光定量PCR具有很高的特异性和敏感性,特别适合样本中微量贝氏柯克斯体DNA的检测,其定量检测可用于动物实验中的贝氏柯克斯体感染程度的分析。     

OmpA-VS2为靶基因的非标记实时荧光PCR检测沙眼衣原体感染

目的建立一种以OmpA-VS2为靶基因的非标记实时荧光聚合酶链反应(PCR)方法,以检测沙眼衣原体。方法设计15种型别沙眼衣原体通用引物,对OmpA-VS2进行套式PCR扩增和实时荧光PCR检测,并对其敏感性、特异性进行评价,并用临床标本进行验证。结果15种型别沙眼衣原体均扩增出168bp的目的片段;敏感性为每个反应1个拷贝。特异性分析可见,与10种泌尿生殖道病原体和共生菌均没有交叉反应。临床标本验证的结果显示,该方法与以质粒为靶基因的核酸扩增检测的Amplicor CT/NG方法的检测结果一致。结论该方法具有敏感性高、特异性强、扩增后无需PCR后处理等特点,可望用于临床与防治项目中沙眼衣原体的检测。     

实时荧光定量PCR检测恙虫病东方体

目的建立检测恙虫病东方体的实时荧光定量PCR(quantitative real-ti me PCR)方法。方法根据恙虫病东方体56kD外膜蛋白基因序列设计引物和探针,以克隆的56kD基因片段作DNA模板,建立实时荧光定量PCR检测方法。结果建立的荧光定量PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.999)。荧光定量PCR检测恙虫病东方体的灵敏度约为套式PCR的100倍,并且具有良好的重复性。用该定量PCR检测其它相关立克次体和病原菌DNA样本,检出结果均为0。用该定量PCR检测恙虫病东方体实验感染小鼠的血、脾脏、肺脏、肝脏标本,结果脾脏中东方体出现最早和检出量最多,肝脏和肺脏次之。血中的恙虫病东方体量较低。结论本研究建立的荧光定量PCR方法具有很高的特异性和敏感性,可用于东方体感染早期血样本的快速检测作恙虫病感染早期诊断,并且可以定量分析评价恙虫病东方体感染的程度。     

一种基于momp基因的衣原体分子生物学甄别技术的初步建立

目的利用PCR方法建立一种基于momp基因的衣原体分子甄别方法。方法根据衣原体momp基因的恒定区和可变区分别设计衣原体科特异性引物和种特异性引物，利用PCR方法对本实验室保存的衣原体进行扩增，以达到甄别的目的。利用限制性片段长度多态性（RFLP）分析科特异性PCR扩增产物，研究momp基因的多态性。结果利用建立的PCR方法对本实验室保存的九株衣原体进行了研究，结果表明八株衣原体都是鹦鹉热嗜衣原体，一株为沙眼衣原体。利用限制性片段长度多态性（RFLP）分析科特异性PCR扩增产物，分析了D34、B11001、CW2和CP12四株衣原体，结果可以产生两种不同的带型。结论利用建立的PCR方法可以达到检测甄别衣原体的目的，并可以通过RFLP分析衣原体的侵袭性。     

嗜水气单胞菌双重荧光定量PCR检测方法的建立与初步应用

目的建立快速准确检测致病性嗜水气单胞菌的双重荧光定量PCR方法。方法针对嗜水气单胞菌的16S rDNA和气溶素基因aerA的序列设计特异性引物及TaqMan探针,优化双重荧光定量PCR反应条件,并结合常规PCR方法及分离培养鉴定对临床样品进行检测和对比验证。结果荧光定量PCR反应体系对质粒标准品的敏感性为10拷贝/反应,是常规PCR检测方法的100倍;该方法检测12种其他种属细菌时未出现假阳性;对实际样品的检测结果其敏感性同样高于普通PCR,定量检测目的基因的拷贝数与样品中目的菌的分离率成正比。结论本方法敏感性高,特异性好,可用于致病性嗜水气单胞菌感染的诊断、疾病防控及流行病学研究。     

犬库布病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立及应用北大核心CSCD

目的建立可检测狐狸物种中犬库布病毒(CaKoV)的SYBR GreenⅠ实时荧光定量PCR检测方法。方法利用PCR方法扩增狐狸CaKoV的3D基因504bp,将其克隆到pEASY-Blunt载体,构建重组质粒作为阳性质粒,以其为模板建立SYBR GreenⅠ实时荧光定量PCR检测方法,验证其灵敏度、特异性和重复性。结果建立的SYBR GreenⅠ实时荧光定量PCR检测方法检测目的基因在6.12×10^(1)~6.12×10^(8)拷贝/μl时呈良好的线性关系,相关系数为0.994,斜率为-4.369,灵敏度6.12×10^(1)拷贝/μl。该方法对于CPV、CDV和CCV未出现特异性扩增。所有标准曲线在溶解温度为86.49℃时出现单一的特异峰。使用本方法检测2021年辽宁、山东地区的253份狐狸临床标本,总阳性率为7.91%。结论建立的CaKoV SYBR GreenⅠ实时荧光定量PCR方法敏感、特异,用于狐狸CaKoV感染的快速检测。     

Development of serodiagnostic kit "HITAZYME Chlamydia Ab" for Chlamydia trachomatis infections using extracted antigen]

To develop EIA kit with low cross-reactivity for the quantitative detection of anti-chlamydial antibodies, we examined the preparation of trachomatis antigens and its specificity to mouse antisera and human sera. The chlamydial elementary body (EB) purified from C. trachomatis L2/434/Bu strain was treated by Sarkosyl, dithiothreitol and SDS by turns to obtain the soluble EB outer membrane (COMC). SDS-PAGE showed that the major components of the COMC were 96K, 60K and 39.5 KDa peptides. The reactivity of the COMC immobilized to 96 wells microtiter plate to mouse anti-serum to C. trachomatis was higher than the other two mouse anti-sera to C. psittaci and pneumoniae. In human sera, the cut off values were calculated from an average optical density plus its two-fold standard deviation obtained by the testing of 100 samples of healthy human sera. We evaluated the specificity of the kit to 17 anti-C. pneumoniae, 9 C. trachomatis and 4 C. psittaci antibodies positive patients' sera judged by the MFA method respectively. The results showed that the concordance ratio of IgG and IgA were 88%, 100% in anti-C. pneumoniae, 89%, 78% in anti-C. trachomatis and 50%, 50% in anti-C. psittaci respectively. From the results obtained in this study, we concluded that the HITAZYME method which had a very low cross-reactivity to C. pneumoniae is clinically useful in the serodiagnosis of C. trachomatis infections, even if it has a little common antigenicity with C. psittaci antigen.     

Detection of Chlamydia trachomatis by polymerase chain reaction]

A polymerase chain reaction (PCR) procedure was developed for detection of Chlamydia trachomatis. Two oligonucleotide primers based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used. A single DNA fragment was amplified, when C. trachomatis DNA was template for the PCR. No amplified product was detected in Chlamydia psittaci DNA, Chlamydia pneumoniae DNA or other bacterial DNAs. The amplified DNA fragment was detected, when DNA of greater than or equal to 10(2) C. trachomatis per reaction was used as template for the PCR. Thus, the PCR was shown to be specific for C. trachomatis and more sensitive than the enzyme immunoassays for detection of chlamydial antigen and the chlamydial rRNA:DNA probe hybridization method.     

羊流产鹦鹉热衣原体感染棘阿米巴的电镜观察

目的　探讨鹦鹉热衣原体能否在自由生活阿米巴中生存繁殖。方法　以阿米巴共培养方法,将羊流产鹦鹉热衣原体分别实验感染多噬棘阿米巴和卡氏棘阿米巴,采用电镜来观察衣原体在阿米巴内的生长情况。结果　电镜显示,鹦鹉热衣原体能够感染卡氏棘阿米巴和多噬棘阿米巴,EB以胞吞的方法进入滋养体内,主要分布于细胞的吞噬体空泡中,并在吞噬体内发育为RB ,以二分裂方式增殖。EB以包涵体的形式释放于细胞体外或细胞裂解的方式直接释放胞外。同时,可见到衣原体EB颗粒在阿米巴包囊内生存。结论　鹦鹉热衣原体能在棘阿米巴内生存和繁殖,环境中自由生活阿米巴可能成为鹦鹉热衣原体的贮存宿主。     

用DNA探针检测肺炎衣原体

目的建立一种敏感而特异的肺炎衣原体分子生物学检测方法。方法用ＰＣＲ扩增约４６０ｂｐ的肺炎衣原体种特异性基因片段并标记成探针,建立ＤＮＡ探针杂交检测肺炎衣原体的方法。结果：探针只与肺炎衣原体ＡＲ－３９、ＶＲ１３１０株ＤＮＡ呈阳性杂交斑点,与肺炎衣原体ＡＲ－３９株全ＤＮＡＰｓｔＩ酶切片段在２．５ｋｂ处有一阳性杂交带,与其它两种衣原体、Ｑ热立克次体、嗜肺军团菌、大肠杆菌、绿脓杆菌、金黄色葡萄球菌ＤＮＡ斑点膜无阳性杂交信号。从１５２例有呼吸道疾患的住院病人鼻咽拭子和肺泡灌洗液中检出阳性１８例,阳性率１２％。结论本文建立的ＤＮＡ探针检测肺炎衣原体方法具有较高的敏感性和特异性,可用于批量临床标本的检测。     

Detection and characterization of Chlamydophila psittaci in asymptomatic feral pigeons(Columba livia domestica) in central Thailand

Objective:To detect and characterize Chlamydophila psittaci(C.psittaci) in asymptomatic feral pigeons in central Thailand.Methods:A total 814 swabs from the trachea and cloacae of 407non-clinical feral pigeons in central Thailand were collected and tested for the presence of C.psittaci.Results:A 10.8%of feral pigeons in the sample group were positive as determined by nested PCR primer specific to C.psittaci.The outer membrane protein A(orupA) gene of positive samples exhibited amino acid identity of C.psittaci ranging from 71 to 100%and were grouped in genotype B.Exceptionally,BF1676-56 isolate was closely related to Chlamydia avium with99%identification of the I6 S ribosomal(r) RNA gene.Conclusions:This is the first report on C.psittaci isolated from asymptomatic feral pigeons in Thailand,which provides knowledge for the disease status in pigeon populations in Thailand.     

鹦鹉热衣原体HSP60基因克隆及其表达产物抗原性的实验研究

目的验证新获得的鹦鹉热衣原体HSP60基因在E.coli中高效表达的产物是否具有足够的抗原活性,为进一步研究其在鹦鹉热衣原体感染检测上的应用奠定基础。方法根据GenBank鹦鹉热衣原体Hsp60基因序列X51404,设计一对特异性引物,扩增了鹦鹉热衣原体HSP60全长基因,克隆入pET32a(+)载体,转化大肠杆菌BL21(DE3)后,利用IPTG诱导获得高效表达。结果扩增了鹦鹉热衣原体HSP60全长基因,表达产物分子量约为68kD,经Western blotting和胶体金免疫层析技术检测,表明表达产物具有较好的抗原性。结论扩增了鹦鹉热衣原体HSP60全长基因,并对其表达产物的抗原性进行免疫印记分析和免疫层析初步检测,表明具有较好的抗原性,为进一步研究其在鹦鹉热衣原体感染检测上的意义打下了基础。     

鹦鹉热衣原体套式PCR和DNA测序方法研究

目的　建立特异、灵敏、快速的鹦鹉热衣原体 (Cps)分子生物学检测方法。 方法　采用套式PCR扩增检测和DNA测序方法 ,并进行实验室评价。结果　 4株Cps均能被套式PCR扩增出 2 14bp目的条带 ,而沙眼衣原体、肺炎衣原体、肺炎支原体等其它微生物均不能检出 ;Cps套式PCR灵敏度高于CpsPCR ;模拟样本均能被检出。Cps(CS株 )套式PCR产物直接测序与Cps典型株序列完全一致。结论　套式PCR是检测Cps特异、灵敏 ,且快速的分子生物学检测方法     

Chest radiograph of atypical pneumonia: comparison among Chlamydia pneumoniae. Pneumonia, ornithosis, and Mycoplasma pneumoniae pneumonia

No report has been found comparing Chlamydia pneumoniae (C. pneumoniae) pneumonia radiographically with other atypical pneumonias, Chlamydia psittaci (C. psittaci) pneumonia and Mycoplasma pneumoniae (M. pneumoniae) pneumonia. We described the chest radiographs of three kinds of pneumonia cases: 46 cases of C. pneumoniae pneumonia, 39 cases of C. psittaci pneumonia, and 131 cases of M. pneumoniae pneumonia. Radiographic shadows were categorized into main shadows and sub-shadows. The main shadows are classified from the viewpoint of the characteristics; air space consolidation(AS), ground-glass opacity(GG), reticular shadow(RS), bronchopneumonia(BP), and small nodular shadows (SN). The size, the site, and the number of the main shadows were also analyzed. In comparison among the three pneumonias, BP was the most frequent in M. pneumoniae pneumonia (0.40/case). AS predominated in C. pneumoniae pneumonia (0.67/case), and GG in C. psittaci pneumonia (0.62/case). The number of main shadows was equal, about 1.4/case in three pneumonias. Large shadows were less frequent in M. pneumoniae pneumonia than C. pneumoniae pneumonia (p = 0.02) and C. psittaci pneumonia (p = 0.01). Main shadows were more frequent in the outer zone in M. pneumoniae pneumonia than C. psittaci pneumonia (p = 0.01), and in the middle zone in C. psittaci pneumonia than in M. pneumoniae pneumonia (p = 0.02). Cases with bilateral main shadows were less common in M. pneumoniae pneumonia (9%) than C. pneumoniae pneumonia(33%, p = 0.001) and C. psittaci pneumonia(30%, p = 0.005). Thickening of bronchovascular bundles as a sub-shadow was most frequently noted in M. pneumoniae pneumonia. Some differences among the three atypical pneumonias were seen in the chest radiograph. However, no specific findings of C. pneumoniae pneumonia were shown radiographically in this study.     

吉林省鸽子鹦鹉热衣原体的分子流行病学调查和基因型分布研究

目的 2015年3-5月调查吉林省长春市和吉林市肉鸽与信鸽中鹦鹉热衣原体的流行情况及基因型分布.方法 本研究共采集鸽子粪便样本399份,其中长春市样本282份,吉林市样本117份.利用PCR技术进行鹦鹉热衣原体ompA基因扩增、测序以及基因型分析.结果 本实验结果显示:鹦鹉热衣原体的感染率为5.01％(21/399),其中吉林市鹦鹉热衣原体的感染率(9.40％)明显高于长春市的感染率(3.19％).此外,品种也是与衣原体感染相关的主要风险因素,肉鸽的感染率为7.49％,而信鸽的感染率为0.ompA基因的序列分析显示,这些鹦鹉热衣原体都属于B型.结论 综上所述,我国吉林省肉鸽具有较高的B型鹦鹉热衣原体流行,给人类的健康带来了潜在的威胁.