应用单一和双重荧光定量PCR法快速检测军团菌

目的 建立单一和双重荧光定量PCR方法分别和同时进行军团菌属及嗜肺军团菌的检测.方法 利用军团菌属16 S rRNA基因和嗜肺军团菌mip基因设计引物和探针,两条基因探针分别标记FAM和HEX,并将相关反应体系和条件进行优化.分别应用单一基因探针(单一荧光定量PCR)和双重基因探针(双重荧光定量PCR)对嗜肺军团菌、非嗜肺军团菌及非军团菌进行检测,并验证两种方法的特异度、敏感度.应用双重荧光定量PCR检测空调水样滤膜样品和DNA提取样品,比较两者结果的一致性.结果 针对军团菌属及嗜肺军团菌,应用荧光定量PCR,16 S rRNA基因和mip基因均能较好的检出,16S rRNA和mip的最低检出限分别为8和10个拷贝.经优化得到了最佳反应体系.单一荧光定量PCR方法所检的8株嗜肺军用菌及4株非嗜肺军团菌16 S rRNA基因均为阳性,嗜肺军团菌mip基因阳性,非嗜肺军团菌mip基因阴性.双重荧光定量PCR方法所检的23株嗜肺军团菌中有2株为假阴性,9株非嗜肺军团菌和非军团菌属中有1株为假阳性.49份空调水样滤膜直接检测和提取DNA后检测的结果一致,其中26份水样军团菌阳性,20份为嗜肺军团菌,6份为非嗜肺军团菌;1份弗朗西斯菌检测HEX阳性(假阳性),占实际培养分离的1/26.结论 单一及双重荧光定量PCR法特异、快速、敏感,一次同时检测嗜肺与非嗜肺军团菌,满足对空调和环境水样军团菌监测的要求.     

应用单一和双重荧光定量PCR法快速检测军团菌

目的 建立单一和双重荧光定量PCR方法分别和同时进行军团菌属及嗜肺军团菌的检测.方法 利用军团菌属16 S rRNA基因和嗜肺军团菌mip基因设计引物和探针,两条基因探针分别标记FAM和HEX,并将相关反应体系和条件进行优化.分别应用单一基因探针(单一荧光定量PCR)和双重基因探针(双重荧光定量PCR)对嗜肺军团菌、非嗜肺军团菌及非军团菌进行检测,并验证两种方法的特异度、敏感度.应用双重荧光定量PCR检测空调水样滤膜样品和DNA提取样品,比较两者结果的一致性.结果 针对军团菌属及嗜肺军团菌,应用荧光定量PCR,16 S rRNA基因和mip基因均能较好的检出,16S rRNA和mip的最低检出限分别为8和10个拷贝.经优化得到了最佳反应体系.单一荧光定量PCR方法所检的8株嗜肺军用菌及4株非嗜肺军团菌16 S rRNA基因均为阳性,嗜肺军团菌mip基因阳性,非嗜肺军团菌mip基因阴性.双重荧光定量PCR方法所检的23株嗜肺军团菌中有2株为假阴性,9株非嗜肺军团菌和非军团菌属中有1株为假阳性.49份空调水样滤膜直接检测和提取DNA后检测的结果一致,其中26份水样军团菌阳性,20份为嗜肺军团菌,6份为非嗜肺军团菌;1份弗朗西斯菌检测HEX阳性(假阳性),占实际培养分离的1/26.结论 单一及双重荧光定量PCR法特异、快速、敏感,一次同时检测嗜肺与非嗜肺军团菌,满足对空调和环境水样军团菌监测的要求.     

诺如病毒遗传组I型TaqMan—MGB探针实时荧光RT-PCR的研究

目的建立诺如病毒遗传组I型TaqMan-MGB探针实时荧光RT-PCR快速、特异、灵敏的检测方法，为疾病预防控制提供可靠的依据。方法根据GenBank诺如病毒遗传组I型代表株保守序列设计特异引物对和TaqMan-MGB探针，建立一步法实时荧光RT-PCR快速检测反应体系，优化反应条件，评价反应体系的灵敏度、特异性、重复性．并与常规RT—PCR比较。结果诺如病毒遗传组I型TaqMan-MGB探针实时荧光RT-PCR检测时限短。仅1h就出结果。与轮状病毒、腺病毒、星状病毒、甲肝病毒、诺如病毒遗传组Ⅱ型无交叉反应，最低检出下限为100拷贝／反应，比常规RT—PCR灵敏100倍，5份浓度不同的诺如病毒遗传组I型标本重复检测5次。平均Ct值变异系数范围为0．39％-1．02％。结论诺如病毒遗传组I型TaqMan-MGB探针实时荧光RT—PCR快速、特异、灵敏、重复性好，可应用于突发公共卫生应急检测和诺如病毒遗传组I型监测，提高快速检测能力。     

荧光定量PCR检测查菲埃立克体

依据查菲埃立克体16SrRNA基因序列设计特异性引物和TaqMan-MGB探针,以克隆的查菲埃立克体16SrRNA基因片段作DNA模板,建立实时荧光定量PCR检测方法。与套式PCR相比较,荧光定量PCR检测的灵敏度是其30倍;用荧光定量PCR检测其他相关立克次体和细菌DNA样本,检出结果为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%~2·0%之间。结果证明本研究建立的荧光定量PCR方法具有种特异性和良好的重复性,可用于检测感染样本中的微量查菲埃立克体DNA。     

马尔堡、埃博拉病毒双重荧光定量PCR检测方法的建立

目的 建立一种快速、敏感、特异的双重实时荧光定量PCR方法,可同时检测马尔堡病毒和埃博拉病毒.方法 通过序列比对挑选出两种病毒基因组中高度保守的序列,分别设计引物及Taqman探针,两条探针分别标记FAM和Texas Red荧光报告基因,建立双重实时荧光定量PCR反应体系.结果 双重荧光定量PCR方法检测两种病毒阳性标准品的灵敏度分别为30.5拷贝/μl和28.6拷贝/μl,通过检测日本脑炎病毒、黄热病毒、登革热病毒无交叉反应,有较好的灵敏度和特异性.结论 建立了马尔堡、埃博拉病毒双重荧光定量PCR检测方法,实现了两种病毒同时实时定量检测,在传染病防控领域有较好的应用前景.     

实时荧光定量PCR检测沙眼衣原体的研究

目的采用TaqMan探针建立检测沙眼衣原体的实时荧光定量PCR（real-time PCR）方法。方法根据沙眼衣原体外膜蛋白A的基因（ompA）序列设计引物和探针,以克隆的ompA部分基因片段作DNA模板,建立实时荧光定量检测方法。结果建立的荧光定量PCR检测方法的最低检出限为5 copies/反应,检测线性范围100～107线性关系良好（r2=0.997）,比巢式PCR敏感100倍;且与鹦鹉热衣原体、淋球菌、解脲脲原体、大肠杆菌等病原菌DNA以及人基因组DNA均无交叉反应,表明该方法具有良好的特异性。以巢式PCR作参比,建立的荧光定量PCR法检测沙眼衣原体的阳性符合率为100.00%,阴性符合率为95.09%,总符合率为96.81%。结论建立的检测沙眼衣原体实时荧光定量PCR具有特异性强和敏感性高的特点,可快速检测样本中微量沙眼衣原体DNA,适用于对沙眼衣原体进行大规模筛选。     

双重实时荧光PCR检测肠出血型大肠埃希菌stx1和stx2基因方法的建立

目的建立一种检测肠出血型大肠埃希菌（EHEC）产毒基因stx1和stx2的TaqMan双重实时荧光PCR法。方法根据GenBank公布的EHECstx1和stx2基因序列进行比对后设计相应的PCR引物和TaqMan探针,探针报告基团分别使用FAM和HEX标记,淬灭基团使用BHQ1标记。建立双重实时荧光PCR反应体系并对反应条件进行优化,对方法的灵敏度、特异性及其重复性进行分析。结果建立了EHEC双重实时荧光PCR检测方法。菌液浓度为102～108cuf/ml时,均可观察到扩增曲线,且扩增曲线平滑;检测30份模拟临床样品,均检测到stx1和stx2基因,且均培养出EHEC,该方法灵敏度为100%;3次重复测定108、105、102cfu/ml3个高、中、低浓度的菌液DNA模板,得到其扩增曲线,计算各浓度的Ct值标准差及变异系数,均小于5%,在误差允许范围内。结论建立的双重实时荧光PCR法可快速、灵敏、特异、有效地检出EHEC。     

肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100％.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.     

反转录酶活性检测实时荧光定量PCR方法的建立

目的 在传统产物增强性反转录酶(PERT)活性检测方法基础上建立检测反转录酶活性的实时荧光定量PCR方法.方法 以噬菌体MS2 RNA为模板,设计、合成引物和探针,并分别以连续10倍倍比稀释的AMV反转录酶(1×10-1～1×10-9 U/μl)标准品催化体外反转录反应合成相应cDNA.采用实时荧光定量PCR法检测底物cDNA模板量,并获得相应的PCR荧光值曲线,分析AMV反转录酶的相对活性.同时应用传统PERT方法对cDNA进行扩增,测定该方法的灵敏度.结果 将AMV反转录酶标准品连续10倍倍比稀释后催化反转录反应,实时荧光定量PCR分析所得的反转录产物cDNA,结果得到与理论值完全相符合的PCR标准荧光值曲线;定量分析结果显示,浓度为1×10-2～1×10-8U/μl的AMV反转录酶均可得到相应的荧光值.不同稀释度的AMV反转录酶,经传统PERT方法扩增后,结果显示浓度为1×10-3～1×10-7 U/μl的AMV反转录酶均可见大小为112 bp的阳性条带,检测灵敏度达到1×10-7U/μl.结论 成功建立了基于实时荧光定量PCR技术的检测反转录酶活性的新方法,实验操作简单,具有高精确性和无污染性,为生物制品外来污染尤其是反转录病毒污染的检测提供了参考指标.     

实时荧光PCR方法快速检测空肠弯曲菌方法的建立

目的建立实时荧光PCR快速检测空肠弯曲菌的方法。方法以空肠弯曲杆菌HipO基因的保守序列为模板设计特异性引物探针,建立一种能快速检测样本中空肠弯曲杆菌的实时荧光PCR方法;对方法的特异性和敏感性进行评价,并以正常人粪便为空白样本,添加一定量空肠弯曲菌标准株菌液进行检测,以对方法的检测效果进行初步评价。结果该实时荧光PCR方法只对空肠弯曲杆菌进行特异扩增,同种属的结肠弯曲菌及其他常见食源性病原菌均不能扩增;整个检测过程只需要80min,对空肠弯曲菌菌悬液可检测至5个细菌,对加标粪便样本可检测至10-100个细菌。结论本研究建立的实时荧光PCR检测空肠弯曲菌方法不仅能实现对空弯菌的快速检测,而且还为空弯菌的快速诊断及其引起的食源性疾病的监控溯源提供有意义的参考。     

Detection of Legionella pneumophila by real-time PCR for the mip gene

A real-time PCR assay for the mip gene of Legionella pneumophila was tested with 27 isolates of L. pneumophila, 20 isolates of 14 other Legionella species, and 103 non-Legionella bacteria. Eight culture-positive and 40 culture-negative clinical specimens were tested. This assay was 100% sensitive and 100% specific for L. pneumophila.     

A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens

Objective: Because presently used methods for diagnosis of Legionella pneumonia lack sufficient sensitivity and sometimes specificity and rapidity, the detection of Legionella spp. by amplification of nucleic acids might be valuable. However, performing polymerase chain reaction (PCR) on clinical samples such as sputum is difficult because of the presence of extraneous DNA and inhibitors of the reaction. An attempt to circumvent these problems was made.
Method: A nested PCR method was devised using primers from the mip gene of Legionella pneumophila. This PCR was tested on pure cultures of legionellae and clinical isolates of other bacteria. Clinical samples (bronchoalveolar lavage fluid, bronchial aspirate and sputum) from patients who suffered from legionellosis and samples from patients who suffered from other causes of pneumonia were also tested.
Results: The PCR was specific for L. pneumophila and no non- Legionella bacteria reacted. Ten to 50 colony forming units of Legionella in the sample could be detected. Twenty-two of 25 clinical samples were positive among patients suffering from pneumonia proven to be due to L. pneumophila serogroups 1, 3, 4, 5 and 6. Two of the three negative samples were from patients who had been treated with adequate therapy for at least 2 days and were culture negative. However, nine other culture-negative samples were PCR positive, of which seven came from patients who had been treated for 3–7 days. All pneumonia patients in the control group proved negative in PCR. A commercial kit for DNA preparation from clinical samples, based on absorption of nucleic acids to silica gel, was superior to the traditional phenol/chloroform extraction and increased the rapidity, simplicity and sensitivity of the procedure.
Conclusions: A nested, simplified and rapid PCR method using mip primers proved to be more sensitive than culture and as sensitive and specific as other PCR procedures previously reported.     

Nucleotide sequence analysis of the Legionella micdadei mip gene, encoding a 30-kilodalton analog of the Legionella pneumophila Mip protein.

After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid pBA6004 revealed a high degree of homology (71%) to the L. pneumophila mip gene, with the predicted secondary structures of the two Mip proteins following the same pattern. Southern hybridization experiments, with the plasmid pBA6004 as the probe, suggested that the mip gene of L. micdadei has extensive homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins).     

Multicenter comparison of molecular methods for detection of Legionella spp. in sputum samples

Legionellosis can be diagnosed by PCR using sputum samples. In this report, the methods of nine laboratories for 12 sputum samples with Legionella pneumophila and Legionella longbeachae are compared. We conclude that (i) liquefaction prevents PCR inhibition, (ii) the employed mip gene PCRs detected L. pneumophila only, and (iii) the 16S rRNA gene PCR detected both Legionella species and is preferred for the diagnosis of legionellosis.     

Identification of mip-like genes in the genus Legionella.

The mip gene of Legionella pneumophila serogroup 1 strain AA100 encodes a 24-kilodalton surface protein (Mip) and enhances the abilities of L. pneumophila to parasitize human macrophages and to cause pneumonia in experimental animals. To determine whether this virulence factor is conserved in the genus Legionella, a large panel of Legionella strains was examined by Southern hybridization and immunoblot analyses for the presence and expression of mip-related sequences. Strains representing all 14 serogroups of L. pneumophila contained a mip gene and expressed a 24-kilodalton Mip protein. Although the isolates of the 29 other Legionella species did not hybridize with mip DNA probes under high-stringency conditions, they did so at reduced stringency. In support of the notion that these strains possess mip-like genes, these species each expressed a protein (24 to 31 kilodaltons in size) that reacted with specific Mip antisera. Moreover, the cloned mip analog from Legionella micdadei encoded the cross-reactive protein. Thus, mip is conserved and specific to L. pneumophila, but mip-like genes are present throughout the genus, perhaps potentiating the intracellular infectivity of all Legionella species.     

Detection of mip gene by PCR for diagnosis of Legionnaires' disease.

The mip gene of Legionella pneumophila was demonstrated by PCR and probing in paired acute-phase and convalescent-phase sera from five patients with Legionnaires' disease but not in the acute-phase sera of 100 patients with pneumonia that showed no serological evidence of Legionella infection. PCR may help in cases presenting diagnostic difficulty.     

Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes

In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Streptococcus pyogenes, simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children (n = 389) and from adults (n = 40). The MB probes and primers are both pathogen specific, namely, the lytA gene for S. pneumoniae, the mip gene for L. pneumophila, and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for S. pneumoniae, 95.8% and 95.4% for H. influenzae, 100% and 100% for S. pyogenes, and 100% and 95.4% for M. pneumoniae, respectively. The sensitivity and specificity for M. pneumoniae relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of C. pneumoniae, the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.     

Legionella pneumophila mip gene sequencing to investigate a cluster of pneumonia cases

AIMS: To use fluorescent antibody (FA) and PCR studies on fixed lung tissue to investigate whether Legionella pneumophila was the cause of pneumonia in a cluster of three haematology patients. METHODS: Cut sections of paraffin blocks of lung tissue were examined by direct FA (DFA) using fluorescently labelled antibody to serogroup 1 and Pontiac strains of L. pneumophila. In addition, a single tube 'hanging drop' nested PCR targeting the mip gene of Legionella was performed on DNA extracted from the lung sections. Products were sequenced using dye terminator chemistry. RESULTS: Numerous fluorescing bacteria were seen on staining with both antibodies in lung tissue from two of the patients. Identical L. pneumophila mip gene sequences were amplified from both DFA-positive lung sections. Two differing L. pneumophila mip sequences were obtained on three separate occasions from the tissue sections from the third patient negative by DFA. These sequences differed slightly from those obtained from the two DFA positive lung tissues. CONCLUSIONS: There is good epidemiological evidence to link the first two cases who had been treated in the same ward prior to development of fever within two days of each other. The significance of results is controversial for the third patient.