A real-time multiplexed PCR assay for rapid detection and differentiation of Campylobacter jejuni and Campylobacter coli

Campylobacter species are the leading agents of bacterial gastroenteritis worldwide. C. jejuni and C. coli together are responsible for more than 95% of all cases of Campylobacter-induced diarrheal disease in the United States. Detection of campylobacteria in clinical samples by conventional culture is problematic and slow due to their complex taxonomy, fastidious growth requirements, and biochemical inertness. The current study describes a rapid, sensitive, and specific real-time polymerase chain reaction (PCR) assay capable of detecting and differentiating C. jejuni (hippuricase gene, hipO) and C. coli (serine hydroxymethyltransferase gene, glyA) in a single reaction, directly from clinical isolates and human feces. The analytical specificity of the assay was demonstrated with a diverse range of Campylobacter species, related organisms, and other diarrhea-inducing bacterial pathogens. The analytical sensitivity of the multiplexed, PCR assay was 10 genome copies for both C. jejuni and C. coli. Following a rapid DNA extraction method (QIAGEN QIAamp DNA stool Mini Kit), the multiplexed PCR identified C. jejuni or C. coli in 100% of fecal samples containing 10(3) colony-forming units (CFU) per gram of feces. This assay represents the first real-time PCR method capable of detecting and differentiating C. jejuni and C. coli in a single reaction.     

新型二聚体突变荧光引物定量检测沙眼衣原体

目的 以二聚体突变荧光引物技术为基础建立对沙眼衣原体进行定量检测的新方法.方法 以沙眼衣原体主要外膜蛋白(MOMP)基因构建重组质粒作为DNA标准品,设计二聚体突变荧光引物,优化定量PCR体系并进行性能评价;同时对148例临床生殖道标本进行检测.结果 建立的二聚体突变荧光引物的定量PCR方法,其线性范围为101～109...     

A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars

Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT detection step involves a DNA enzyme immunoassay (DEIA) using probes for serogroups (group B, C, and intermediate) and the cryptic plasmid, permitting sensitive detection of 19 Ct serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) without any cross-reactivity with other Chlamydia species and pathogenic bacteria or commensal organisms of the genital tract. Ct-positive samples are analyzed by a nitrocellulose-based reverse hybridization assay (RHA) containing probes for the 19 different serovars and for the cryptic plasmid. The sensitivity of the PCR-DEIA on clinical specimen is equivalent to that of the Cobas TaqMan assay [kappa = 0.95 (95% confidence interval = 0.92 to 0.99)]. Using the RHA, 98% of the Ct-DT detection step-positive samples could be typed. Analysis of cervical swabs from Uganda and The Netherlands revealed that the most common serovars in Uganda are G/Ga (45%), E (21%), K (13%), and F (8%), and in The Netherlands serovars E (38%), F (23%), G/Ga (11%), and D/Da (7%) were most common. Thus, multiplex broad-spectrum PCR in combination with DEIA and RHA permits highly sensitive and specific detection and identification of Ct serovars.     

Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis and Ureaplasma parvum

ObjectivesChlamydia trachomatis and Ureaplasma urealyticum are common pathogens of sexually transmitted diseases. The majority of human ureaplasma isolates belong to the new species U. parvum. Clinically, C. trachomatis and U. parvum usually double infect in the nongonococcal urethritis patients. A novel method for simultaneous detection of C. trachomatis and U. parvum was set up in the present work.Design and methodsMultiple real-time quantitative PCR was developed to allow for rapid, sensitive, specific and quantitative detection of C. trachomatis and U. parvum, simultaneously. To evaluate the applicability of the multiplex real-time quantitative PCR assay to clinical specimens, 64 samples of cervical swabs collected were studied.ResultsCompared to the results obtained from single real-time quantitative PCR of C. trachomatis and U. parvum, the specificity, sensitivity and quantitative detection results of multiple real-time quantitative PCR are approximately identical with those of the former.ConclusionsThis assay will be of great value in the simultaneous and rapid diagnosis of C. trachomatis and U. parvum in the future.     

Duplex real-time PCR assay for rapid detection of ampicillin-resistant Enterococcus faecium

Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the D-Ala-D-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.     

PCR detection and differentiation of Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia trachomatis.

A PCR-based system was developed for the detection and differentiation of Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae. A conserved 145 bp fragment of the chlamydial omp1 gene was amplified from all three species. The three species were then differentiated from each other by digestion of this PCR product with restriction enzymes Eco RI and either Hind III or Pst I. The system was shown to work for two strains of C. pneumoniae, 11 strains of C. psittaci and 10 serovars of C. trachomatis, and had a sensitivity of less than 10 chlamydial elementary bodies. This method was also applicable to the detection of C. trachomatis in conjunctival and nasopharyngeal swabs.     

实时荧光定量-聚合酶链反应方法检测肺炎支原体

目的 建立一种快速、灵敏、特异的肺炎支原体实时荧光定量-聚合酶链反应(real-time PCR)检测方法,以期用于临床肺炎支原体感染检测。 方法 通过测序分析和序列比对,选取肺炎支原体p1基因中保守区域设计特异性引物和荧光探针,建立和完善此real-time PCR检测方法,并进行扩增效率、灵敏度及特异度评价。与已报道的肺炎支原体常规聚合酶链反应(PCR)方法进行150份临床标本检测能力比较。 结果 建立的real-time PCR方法对肺炎支原体的检测限约为10 cfu。使用该方法对9株肺炎支原体ATCC标准株和30株临床分离株核酸扩增均为阳性;对10种其他支原体、13种常见呼吸道病原菌染色体及人类染色体扩增结果均为阴性。同时,临床标本的检测结果显示该方法检测灵敏度优于常规PCR。 结论 本研究建立的real-time PCR方法可快速、灵敏、特异地检测标本中肺炎支原体核酸,可适用于临床肺炎支原体诊断。     

Enzyme-linked immunosorbent assay for the detection of antibody to Chlamydia trachomatis and Chlamydia psittaci

An enzyme-linked immunosorbent assay (ELISA) was developed with use of a strain of Chlamydia trachomatis, lymphogranuloma venereum serotype 2. The ELISA reactions were monitored by absorbance at 492 nm in a spectrophotometer. A positive control method based on one serum dilution was used to assign ELISA titers to the sera. Sera that had been tested with use of complement fixation (CF) and microimmunofluorescence (MIF) tests were examined with ELISA. ELISA was more sensitive than the CF test and as sensitive as the MIF test. Serum pairs that had diagnostic titer rises in the other tests also had such rises in ELISA. ELISA is a simple, sensitive assay for detection of antibody to C. trachomatis.     

Development of a multiplex TaqMan real-time PCR assay for the detection of Chlamydia psittaci and Chlamydia pneumoniae in human clinical specimens

Diagnosis of Chlamydia psittaci and Chlamydia pneumoniae infections has traditionally relied on serological assays. We developed a multiplex real-time PCR assay for detection of C. psittaci, C. pneumoniae and an internal control. Results of this assay demonstrated 100% concordance compared to results of previously tested human clinical specimens.     

A PCR assay for rapid detection of vancomycin-resistant enterococci

Since the first report of a vancomycin-resistant enterococcal clinical isolate, these Gram-positive bacteria have emerged as important nosocomial pathogens. Several glycopeptide resistance phenotypes can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin. In the present study, we developed a multiplex PCR, which allows the simultaneous identification of enterococci at the genus level and detection of the most frequent glycopeptide resistance genotypes. Five primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 and tuf were used in one reaction tube with bacterial DNA extracted from three to five colonies. This PCR method is suitable for the rapid detection of vancomycin-resistant enterococci.     

A TaqMan-based quantitative real-time PCR assay for identification of the goose circovirus

Goose circovirus (GoCV) is a potential immunosuppressive virus that poses a great hazard to the goose industry and has been shown to be widely distributed throughout China. We have established a fast, sensitive and highly specific TaqMan real-time quantitative PCR detection method for this virus. Specific primers and probes were designed against the conserved regions of the genomic GoCV Rep gene. The results showed that the assay was highly specific and sensitive for GoCV and did not cross-react with other non-targeted waterfowl viruses. The established method will be helpful for epidemiological detection and may be effective in the prevention and control of the disease.     

A real-time PCR assay for the detection of Neisseria gonorrhoeae by LightCycler

The detection of Neisseria gonorrhoeae by the polymerase chain reaction (PCR) is now recognized as a sensitive and specific method of diagnosing infection by the organism. In this Study 152 urine specimens were examined for N. gonorrhoeae by a real-time PCR method using the LightCycler platform and results were compared to an "in-house" PCR assay using an ELISA-based detection method. N. gonorrhoeae DNA was detected in 29 (19%) specimens by LightCycler PCR (LC-PCR) and in 31 (20%) specimens by the "in house" PCR method. The LightCycler assay proved to be specific and 94% sensitive when compared to the "in house" PCR method. These features combined with the rapid turn-around time for results makes the LC-PCR particularly suitable for the detection of N. gonorrhoeae in a routine clinical laboratory.     

Multicolour,multiplex real-time PCR assay for the detection of human-pathogenic poxviruses

After eradication of variola virus, one of the most dangerous infectious diseases affecting mankind, today other poxviruses of different genera can cause infection in humans. These viruses include human-specific molluscipoxviruses as well as zoonotic orthopoxviruses and parapoxviruses. While non-variola orthopoxvirus infections mostly cause mild symptoms in immunocompetent persons, they can evoke severe disease in immunocompromised patients. Since the typical poxviral skin lesions are rarely diagnosed by physicians, PCR-based identification of suspected poxviruses is often required. To simplify the PCR-based diagnosis of human-pathogenic poxviruses, we established a multicolour multiplex real-time PCR that simultaneously detects and differentiates human-pathogenic poxviruses in one reaction. Using 5′ nuclease probes labelled with FAM for orthopoxviruses, VIC for parapoxviruses and FAM and VIC for molluscipoxviruses, respectively, amplification of poxviral DNA resulted in a genus-specific reporter-dye profile. Validation with 36 human clinical specimens and DNA of pathogens causing pox-like skin lesions demonstrated the specificity of the assay. Probit analysis revealed a limit of detection of 9.7, 22.08 and 28.1 copies/assay (95% CI) for molluscipoxvirus, orthopoxvirus and parapoxvirus DNA, respectively. The combinatorial multicolour strategy applied has the potential to be used in further applications targeting even more than three pathogens.     

产毒型霍乱弧菌实时定量三重TaqMan PCR快速检测方法的建立

应霍乱弧菌产生霍乱毒素的能力.     

A real-time PCR assay for detection of emerging infectious Elizabethkingia miricola

Elizabethkingia miricola, a Gram-negative bacillus, is emerging as a life-threatening pathogen in both humans and animals. However, no specific and rapid diagnostic method exists to detect E. miricola. Here, we established a real-time PCR assay for the rapid, sensitive, and specific detection of E. miricola with a wide dynamic range of 10⁸ copies/μL to 10² copies/μL. The detection limit of the real-time assay was 145 copies/μL, which was 100 times more sensitive than conventional PCR. All clinical isolates E. miricola from different host species yield very close Tm (80.25 ± 0.25 °C). Additionally, no cross-reaction or false positives were observed in the assay for non-target bacterial species. The performance of this assay was primarily assessed by testing frog tissue samples. Overall, our study provided a real-time PCR assay, which is a rapid, sensitive, and specific diagnostic method that could be used for early diagnosis and epidemiological investigation of E. miricola.     

应用TaqMan实时荧光-聚合酶链反应方法快速检测粪便标本空肠弯曲菌的研究

目的建立空肠弯曲菌TaqMan实时荧光-PCR方法,用于粪便标本的直接检测。方法根据空肠弯曲菌特异性基因hipO和mapA分别设计引物和探针,在对2组引物和探针进行灵敏度、特异性和重复性评价的基础上,对45例临床腹泻患者粪便标本提取DNA之后,荧光PCR检测,同时进行分离培养。 结果两组引物和探针能准确检测空肠弯曲菌菌株2株,检测限可达到10~20 cfu/ml,并与其他肠道致病菌无交叉反应。检测45份腹泻病例粪便标本,该方法检测到3份为阳性,同时进行的传统培养方法仅从该3份标本中的两份中分离到空肠弯曲菌。 结论本研究建立的TaqMan荧光PCR检测粪便标本中所携带的空肠弯曲菌灵敏度高,特异性好,能够提高粪便中空肠弯曲菌的阳性检出率和缩短检测时限。     

Ultra-rapid detection of Chlamydia trachomatis by real-time PCR in the LightCycler using SYBR green technology or 5'-nuclease probes

Chlamydia trachomatis infections are among the most common sexually transmitted diseases and of great epidemiological importance world-wide. Identification of this pathogen can be difficult, and it is highly desirable to have a rapid and accurate nucleic acid based detection method. Several commercial PCR test systems are available (e.g. CobasAmplicor, Roche, Mannheim, Germany) but they require post-amplification detection by hybridization resulting in extended work-up time and possible cross-contamination. The objective of our study was to develop a routine diagnostic method for the sensitive, specific and rapid detection of C. trachomatis. The obvious choice is real-time PCR without any post-amplification procedures. The dye SYBR Green I (intercalating in dsDNA) provides a simple and fast real-time PCR in the LightCycler. Specific primer design combined with melting curve analysis allows a reliable and sensitive identification of C. trachomatis. In addition, a new commercial real-time PCR system (RealArt C. trachomatis LC PCR Reagents, artus, Hamburg, Germany) was evaluated, that combines sequence-specific primers and fluorescence-labelled (FRET) 5'-nuclease probes. An internal control integrated in this system detects false negative results and erroneous PCR conditions. All results were compared with the corresponding data from an analysis using the CobasAmplicor system (Roche). (Clin