Evaluation of genus-specific and species-specific real-time PCR assays for the identification of Brucella spp.

BACKGROUND: The identification of Brucella isolates using conventional microbiological techniques is time-consuming and hazardous. We therefore assessed the performance of real-time PCR assays for the identification of members of the genus Brucella to the genus and species level. METHODS: We evaluated an in-house developed assay and various previously published real-time PCR assays targeting bcsp31, per, IS711, alkB/IS711 and BMEI1162/IS711 using 248 Brucella strains representing the biotypes of all species and a large panel of clinically relevant, phylogenetically related and serologically cross-reacting bacteria. RESULTS: No misidentification was observed. However, several biotypes of Brucella abortus and Brucella suis were not detected with some of the published assays. The limit of detection varied widely among the assays (16-1600 fg) demonstrating that some assays should not be applied to clinical samples but may help to identify colony material. CONCLUSIONS: In summary, most of the assays revealed low detection limits and proved to be highly selective for the detection of the genus Brucella and the species that are most relevant for humans. Assays targeting the bcsp31 gene can be recommended to screen for Brucella. Species-specific assays should be consecutively applied confirming the primary diagnosis by a second gene target.     

Development and evaluation of real-time polymerase chain reaction assays on whole blood and paraffin-embedded tissues for rapid diagnosis of human brucellosis

This study aimed at developing a real-time polymerase chain reaction (PCR) assay for the rapid diagnosis of human brucellosis on clinical specimens. Three assays with hybridization probe detection on the LightCycler instrument were developed and compared targeting the 16S-23S internal transcribed spacer region (ITS) and the genes encoding for omp25 and omp31. All assays showed 100% analytical sensitivity and 100% specificity when tested on 28 consecutive clinical isolates of Brucella sp. and 19 clinical isolates of common Gram-negative and Gram-positive bacterial pathogens, respectively. The ITS assay was the most sensitive with a limit of detection of 2 genome equivalents per PCR reaction. This assay was then clinically validated prospectively with 354 samples (351 whole blood samples and 3 paraffin-embedded tissues) collected from 340 patients, 24 samples from patients with active brucellosis including 2 relapsing cases, 31 with treated brucellosis, and 299 seronegative patients where brucellosis was initially suspected. The clinical sensitivity, specificity, and positive and negative predictive values of the ITS assay were 66.7%, 99.7%, 94.1%, and 97.6%, compared with culture at 77%, 100%, 100%, and 97.3%, respectively. The difference in sensitivity between culture and ITS-PCR was not statistically significant (P = 0.71). Both relapsing cases were PCR positive. Treated patients were PCR negative. All 3 assays were positive on tissue samples, but the omp25 and omp31 assays did not detect Brucella sp. DNA in blood samples. Because omp31 is not present in Brucella abortus, these data indicate that the 28 tested isolates are most likely Brucella melitensis. ITS-PCR is rapid and could augment the clinical laboratory diagnosis of human brucellosis.     

Loop-mediated isothermal amplification assay targeting the omp25 gene for rapid detection of Brucella spp

A novel loop-mediated isothermal amplification (LAMP) assay was established to detect Brucella species DNA in milk and blood samples of animals and humans. This LAMP assay based on the sequence of highly repetitive omp25 gene was able to detect 9fg/μl Brucella spp. DNA with high sensitivity, which was 10 times higher than the nested PCR. The LAMP was evaluated for its specificity using 19 strains of six Brucella species and 28 related non-Brucella micro-organism strains as controls. The target 19 Brucella strains were all amplified, and no cross-reaction was found with all the non-Brucella micro-organism strains. Both nested PCR and LAMP assays were then used to detect Brucella spp. DNA in 78 milk samples and 113 blood samples from animals and 11 blood samples from humans, and the established LAMP assay yielded 99.0% concordance rate with the nested PCR. The LAMP assay should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of Brucellosis.     

布鲁菌PCR快速检测方法的建立及应用

目的建立布鲁菌聚合酶链反应(PCR)方法,对其进行应用评价。方法针对布鲁菌外膜蛋白编码基因(omp-2)设计PCR引物,建立PCR方法,利用19种常见细菌和布鲁菌株DNA分别对其进行特异性和敏感度评价,对3株疑似布鲁菌株进行核酸检测,并与分离培养法进行结果比对。结果本研究建立的布鲁菌PCR检测方法仅能检出布鲁菌阳性菌株,对照菌DNA未出现目的条带;敏感度为100拷贝/反应;PCR方法对3株疑似布鲁菌株检出omp-2基因条带,鉴定结果为布鲁菌,与分离培养法结果相同。结论本研究建立了一种布鲁菌PCR检测方法,与分离培养法相比,布鲁菌PCR方法准确、快速,适合布鲁菌病疫情的快速检测。     

Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis

Real-time PCR-based assays specific for Brucella abortus, Brucella melitensis and Brucella suis were developed. The assays utilize an upstream primer that is derived from 3' end of the genetic element IS 711, whereas the downstream primers and probes are designed from signature sequences specific to a species or a biovar. The PCR reactions were monitored for fluorescence resonance energy transfer by including two adjacent labeled probes that hybridize to the amplicons as they are formed. The upstream probes were labeled with fluorescein at 3' end while Cy5 was attached to the 5' end of the downstream probes. An increase in the ratio of fluorescein to Cy5 fluorescence during the cycling was indicative of positive amplification event. The assays were accomplished in less than 30 min using a LightCycler in real-time mode. The assays were tested on known strains as well as field isolates and were found to be specific for all known biovars of B. abortus, B. melitensis and biovar 1 of B. suis. Therefore, specificity, sensitivity, speed and real-time detection make these assays attractive for use in epidemiological and ecological studies.     

Single-run, parallel detection of DNA from three pneumonia-producing bacteria by real-time polymerase chain reaction

A molecular assay for parallel detection of three bacteria, Chlamydia (C.) pneumoniae, Legionella (L.) spp., and Mycoplasma (M.) pneumoniae, in clinical specimens by a set of real-time polymerase chain reactions (PCRs) in a single run was evaluated. Bacterial DNAs were extracted by an automated DNA extraction protocol on the MagNA Pure LC System. Amplification and detection were done by real-time PCR on the LightCycler (LC) instrument. For amplification, specific oligonucleotides derived from the 16s rRNA genes of C. pneumoniae, L. spp., and M. pneumoniae were used. The three assays were complemented with an internal control (IC), a specially designed DNA fragment which contains the specific primer binding sites for the three PCRs. The IC was added to the samples, co-extracted, and co-amplified. Primers and hybridization probes were designed to suit one LC PCR program. LC PCRs were established, detection limits were determined, and clinical samples were tested. The detection limits were found between 5.0 and 0.5 IFU/CFU per PCR reaction for each of the bacteria. A total number of 100 clinical specimens were tested for validation of the molecular assay. Tested samples included 63 bronchoalveolar lavages (BALs) and 37 induced sputa specimens. The internal control was detected in all negative and low-positive samples; no inhibition was found throughout the whole study. Additionally, samples underwent testing by culture for L. spp., and M. pneumoniae; for C. pneumoniae, the serological microimmunofluorescence (MIF) test was used. In conclusion, the developed set of LC PCR assays permits parallel detection of C. pneumoniae, L. spp., and M. pneumoniae in a single LC run. This molecular assay may lead to accurate and early diagnosis of pneumonia produced by these three types of bacteria. The assay proved to be suitable for the high-throughput routine diagnostic laboratory.     

SYBR GreenI染料法定量PCR检测疟原虫感染及鉴别虫种的临床应用

目的评价一种可以快速定量检测疟原虫及虫种鉴别的荧光定量PCR法。方法将SYBRGreenI法与TaqMan探针法检测疟原虫进行比对,评价定量的准确性及方法的敏感度。以PCR-DNA测序法为金标准,评价SYBRGreenI法分型的准确性。结果两个方法的定量拷贝检测数无显著性差异（P〉0．05）。SYBRGreenI法检测疟原虫的最低检测下限为1×10^2copy／mL至5×10^2copy／mL之间。与PCR-DNA测序法比较,SYBRGreenI法能准确的对感染的疟原虫虫种做准确的鉴定。结论SYBRGreenI染料法定量可同时进行人体疟原虫的定量检测和虫种鉴别,且成本低廉,操作简便快速,可以在疟疾的高发地区推广使用。     

Development and evaluation of dual-target real-time polymerase chain reaction assays to detect Bordetella spp

Novel, highly specific, and sensitive real-time polymerase chain reaction (PCR) assays using 2 targets, insertion sequence (IS481) and pertussis toxin subunit 1 (ptxS1), were developed to detect Bordetella pertussis and to differentiate between relevant Bordetella spp. Sixty-four non-Bordetella isolates were negative by both assays, demonstrating the specificity of the assays. B. pertussis, Bordetella parapertussis, and Bordetella holmesii isolates were specifically identified using the assays. The lower limit of detection was less than 10 genomic equivalents per reaction for the IS481 and ptxS1 assays. These assays were evaluated using 145 human clinical specimens obtained during cough-illness outbreak investigations, and PCR results were compared with Bordetella spp. culture results. Twenty-seven (18.6%) specimens had late positive cycle threshold (Ct) values (35 相似文献   

Diagnostic value of real-time capillary thermal cycler in virus detection

Molecular-based detection methods such as PCR techniques have had a significant impact on the diagnosis of viral infections because of their superior sensitivity and rapid turnaround time. This review describes the use of real-time PCR on the capillary thermal cycler, the Roche LightCycler trade mark, for early disease detection in diagnostic virology. The advantages of using the LightCycler, the detection processes using SYBR Green I and different hybridization strategies will be discussed in detail, with specific examples drawn from our in-house viral assays. The use of the LightCycler for the investigation of two recent viral outbreaks in Singapore will also be briefly described.     

6种食品致病菌的多重PCR检测

目的寻求食物中毒诊断及食品检测的有效方法。方法建立多重PCR体系一次检测多种食品致病菌方法。结果可以一次检测出肠毒性大肠埃希菌(E.coli-ETEC),蜡样芽胞杆菌(Bacilluscereus),沙门菌属(Salmonellaspp),大肠埃希菌O157:H7(E.coli-O157:H7),志贺菌属(Shigellaspp),霍乱弧菌(Vibriocholerae)等菌属或菌。结论多重PCR体系检测6种菌DNA的灵敏度最低为10.30fg,与单独的PCR检测的灵敏度相同。将它做成体外基因诊断试剂盒,将成为细菌性食物中毒诊断及食品检测的有效工具。     

Techniques actuelles de diagnostic des infections virales respiratoires en réanimation

Hundred viruses can be isolated in patients suffering from respiratory virus infections and hospitalised in intensive care unit (ICU): influenza virus, respiratory syncytial virus, para-influenza virus, adenovirus, coronavirus, rhinovirus, enterovirus, human metapneumovirus, bocavirus… Nasal or tracheobronchial specimens, which contain many epithelial cells will be used to isolate these common viruses. In immunocompromised patients a bronchoalveolar lavage has to be added to these specimens in order to detect cytomegalovirus and some adenovirus. The immunofluorescence or immunoenzymatic assays, which detect viral antigens in the infected cells are the easiest and fastest diagnostic methods, theoretically. As with other techniques, specimen quality is a major determinant of their performance. Unfortunately, the sensitivity of the antigen detection assays is low in respiratory infections in adults. Then the virus recovery by cell culture, which is usually more sensitive than the antigen detection assays, can be helpful. Many studies have reported more respiratory virus detections using nucleic acid testing such as PCR. They detect viruses, which are missed by conventional methods and increase the detection of common respiratory virus. Multiplex PCR assays have been developed, and these can simultaneously detect several viruses directly in clinical specimens. Nucleic acid testing can subtype viruses using subtype-specific primers, and analyse strain variation through genetic. It can be used also to quantify the viral load in clinical specimens. More recently real-time RT-PCR assays have been developed to get more rapidly the results of the nucleic acids assays. Specimen quality, timing and transportation conditions may be less critical for nucleic acid testing than for culture or antigen detection, as viable virus and intact infected cells need not to be preserved. Moreover, viral nucleic acids are detectable for several days longer into the clinical course than is cultivable virus, potentially allowing a diagnosis to be made in late-presenting patients. However, in a clinical virology laboratory, where the speed, low cost, and high sensitivity of the methods are required, the sequential use of antigen detection tests and multiplex PCR could be the best choice, particularly in the clinical setting of respiratory virus infections in adults hospitalised in ICU. In the future, the development of real-time multiplex PCR is likely to be top-priority.     

Development and comparative evaluation of real-time PCR and real-time RPA assays for detection of tilapia lake virus

Tilapia lake virus (TiLV) is a newly emerged pathogen responsible for high mortality and economic losses in the global tilapia industry. Early and accurate diagnosis is an important priority for TiLV disease control. In order to evaluate the methodology in the molecular diagnosis of TiLV, we compared newly developed quantitative real-time PCR (qPCR) and real-time recombinase polymerase amplification (real-time RPA) assays regarding their sensitivities, specificities and detection effect on clinical samples. Real-time RPA amplified the target pathogen in less than 30 min at 39 °C with a detection limit of 620 copies, while qPCR required about 60 min with a detection limit of 62 copies. Both assays were specific for TiLV and there were no cross-reactions observed with other common fish pathogens. The assays were validated using 35 tissue samples from clinically infected and 60 from artificially infected animals. The sensitivities for the real-time RPA and qPCR assays were 93.33 and 100%, respectively, and the specificity was 100% for both. Both methods have their advantages and can play their roles in different situations. The qPCR is more suitable for quantitative analysis and accurate detection of TiLV in a diagnostic laboratory, whereas real-time RPA is more suitable for the diagnosis of clinical diseases and preliminary screening for TiLV infection in poorly equipped laboratories as well as in fish farms.     

Laboratory-based diagnosis of brucellosis--a review of the literature. Part I: Techniques for direct detection and identification of Brucella spp

Brucellosis is a world-wide re-emerging zoonosis and the most frequent laboratory-acquired bacterial infection, causing severe disease in humans with unspecific clinical signs affecting numerous organs. Contact with infected animals, ingestion of contaminated animal products and handling of Brucella isolates in laboratories are risk factors. Various other febrile illnesses, e.g. malaria, tuberculosis, typhoid fever and tularemia may present with the same symptoms. Therefore, clinical diagnosis is difficult to establish but effective therapy requires an early diagnosis. Vaccines for humans are still not commercially available. Blood culturing of Brucella is time-consuming and not reliable. Thus diagnosis is usually based on indirect serological tests, i.e. serum agglutination test, complement fixation or the Coombs test. However, these 'conventional' serological tests lack sensitivity and specificity. Hence, a combination of various tests is mandatory for a definite diagnosis. Enzyme-linked immunosorbent assays can be used for screening and confirmation of brucellosis in one step. Molecular techniques like the polymerase chain reaction and restriction fragment length polymorphism are needed to differentiate species and strains within the genus Brucella. This review will summarize advantages and disadvantages of the techniques used in clinical laboratories for direct detection and identification of Brucella spp.     

Detection of aacA-aphD, qacEδ1, marA, floR, and tetA genes from multidrug-resistant bacteria: Comparative analysis of real-time multiplex PCR assays using EvaGreen(®) and SYBR(®) Green I dyes

We have developed multiplex real-time PCR assays that utilize DNA-intercalating dyes, SYBR Green I (SG) and EvaGreen (EG), with two primer sets (set 1=qacEδ1, tetA and aacA-aphD; set 2=tetA, marA, and floR) to simultaneously amplify the qacEδ1, tetA, aacA-aphD, marA, and floR genes. Validity of the multiplex PCR assays was confirmed by testing 83 bacterial isolates, including Staphylococcus aureus (28 isolates), Enterococcus spp. (17 isolates), Salmonella enterica serovar Typhimurium (8 isolates), Citrobacter spp. (9 isolates), Escherichia coli (14 isolates) and Aeromonas veronii (7 isolates), and performing sequence analysis of representative PCR products. Agarose gel analysis revealed the presence of correct size PCR products, and the differences in their thermal melting (T(m)) curves were used to distinguish various PCR products. Although T(m) peaks of different amplicons after EG-based singleplex and multiplex PCR assays were resolved nicely, only one or two peaks were seen for SG-bound amplicons. EG-based multiplex real-time PCR assays provided better peak resolution. There was a good correlation with a better linear relationship between the C(t) and log input DNA concentration for the set 1 and set 2 genes in EG-based assays (R(EG)(2)=0.9813and0.9803) than in SG-based assays (R(SG)(2)=0.5276and0.6255). The sensitivities of detection were 2.5-25fg and 25-250fg of template DNA in EG and SG-based singleplex and multiplex PCR assays, respectively. The assays, which could be completed in less than 45min, offer sensitive and rapid detection of qacEδ1, aacA-aphD, marA, floR, and tetA genes from a diverse group of multiple antibiotic-resistant bacterial strains.     

Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira

Leptospirosis is an emerging infectious disease, which is considered to be the most widespread zoonotic disease in the world. There are more than 230 known serovars in the genus Leptospira. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic Leptospira spp. was developed and evaluated through amplification of the lipL41 gene coding for the outer membrane protein LipL41. The LAMP assay did not rely on the isolation and culture of leptospires, and no cross-reactivity was observed with other bacterial species. A SYBR Green I-based LAMP assay was also carried out for the real-time detection of DNA amplification. The lower detection limit of the LAMP assay was approximately 100 copies, which was the same as the polymerase chain reaction (PCR) and real-time PCR assays. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis of the amplified product. The LAMP assay is easy to perform and inexpensive, and so may be applied in the rapid and specific diagnosis of Leptospira.     

Diagnosis of human brucellosis using PCR

Brucellosis is a zoonosis caused by facultative intracellular bacteria of the genus Brucella, which are widely distributed in both humans and animals, especially in the developing world. The diagnosis of human brucellosis requires isolation of the bacteria or confirmation through serologic tests. However, culture sampling sensitivity is often low, depending on the disease stage, Brucella species, culture medium, quantity of circulating bacteria and blood culture technique employed. The development of the PCR has offered a new dimension in the diagnosis of different microorganisms, which is possible in just a few hours. Over the past decade, there have been major advancements in all aspects of molecular diagnostics with regard to human brucellosis. PCR-based tests are proving to be faster and more sensitive than traditional methods. However, the sensitivity and specificity of the PCR for Brucella vary between laboratories and no standardization of sample preparation, target genes and detection methods have been established yet. Therefore, in this study, all the important aspects of the PCR for Brucella DNA detection and its utility in the diagnosis and follow-up of patients with brucellosis are reviewed.     

基于M1磁珠富集血液中念珠菌快速检测方法的建立

  目的  建立3种基于重组人甘露聚糖结合凝集素（MBL）蛋白（M1蛋白）磁珠富集技术的血液微量念珠菌快速检测方法（荧光定量PCR法、微培养法、铺板培养法）。  方法  根据荧光定量PCR方法的扩增效果,比较3种裂解液（酵母裂解液、LSTE和TXTE）提取微量念珠菌DNA的效果;通过检测液体培养后的菌量,比较5种微培养液（PBS、SA、YPD、LB和BHI）的增菌效果;使用人血浆模拟样本评价建立的3种方法和标准血培养法对血液中微量念珠菌的检测效果。  结果  与TXTE和LSTE相比,酵母裂解液提取微量念珠菌DNA的效果最佳。 SA、YPD、LB和BHI 4种微培养液中的菌量随时间延长均有上升趋势,特别是BHI增菌效果最佳。 人血浆模拟样本中,荧光定量PCR法虽然能够在最短时间内（3.75 h）检测到病原菌,但不能检出全部阳性样本。 与传统血培养法相比,微培养法和铺板培养法可分别提前至少39 h和22 h获得检测结果,灵敏度为10菌落形成单位/mL（CFU/mL）。  结论  与传统血培养法相比,荧光定量PCR法、微培养法和铺板培养法均能有效缩短检测时间,并且有较高的特异性和灵敏度。     

小片段PCR产物对布鲁菌种属鉴定的研究

目的 应用小片段PCR产物对布鲁菌进行快速种属鉴定.方法 收集2008年1月至2009年6月在吉林省松原地区从92例布鲁菌病患者血液中分离出的布鲁菌13株,其中羊布鲁菌9株,牛布鲁菌2株,猪布鲁菌2株.根据布鲁菌属细菌重复插入序列IS711及羊布鲁菌、牛布鲁菌及猪布鲁菌种间特异性基因片段设计引物,对3株布鲁菌标准菌株及13株临床分离布鲁菌分别进行PCR反应,获得相应PCR产物.将PCR产物T-A克隆并进行测序分析.对布鲁菌标准菌株DNA模板进行稀释,对每一稀释度的DNA模板分别进行10次PCR扩增,进行灵敏度和稳定性检测.结果 4种PCR反应均可获得特异的小片段PCR产物,片段长度分别为63、67、81和83 bp,T-A克隆测序结果 与目的 基因片段比较,符合率为100%,检测模板的最低DNA浓度为1 μg/L,分别用不同的引物对相应布鲁菌DNA模板进行10次PCR扩增,均得到预期结果 .结论 本研究建立的PCR反应能鉴定布鲁菌种属,具有良好的特异性、灵敏度和稳定性.     

Development of BruAb2_0168 based isothermal polymerase spiral reaction assay for specific detection of Brucella abortus in clinical samples

Bovine brucellosis, predominantly caused by Brucella abortus is one of the most neglected zoonotic diseases causing severe economic losses in the dairy industry. The early and precise diagnosis of the disease is required to reduce the transmission of infection in humans as well as animals. In the current study, a rapid and novel isothermal amplification-based polymerase spiral reaction (PSR) was developed for the specific detection of Brucella abortus by targeting the BruAb2_0168 gene. The assay could be conducted at 65 °C in a water bath and results can be obtained after 60 min. The detection limit of the PSR assay was found to be 1.33 fg. The sensitivity of the assay was found to be 10⁴ fold higher than conventional PCR and equivalent to real-time PCR (RT-PCR). The assay didn't exhibit cross-reaction with selected pathogenic non-Brucella bacteria and Brucella spp. other than B. abortus. Forty clinical samples were also tested using this novel assay and it was able to detect 25 samples as positive, however, conventional PCR could detect the targeted organism in 22 samples only.To the extent of our knowledge, this is the first report towards the development of a PSR assay for specific detection of B. abortus. The assay can be used as a quick, sensitive and accurate test for the diagnosis of bovine brucellosis in the field setting. Relatively one of the paradigm-shifting aspects of this assay would be it does not require any expensive equipment and the results can be easily visualized by the unaided eye, therefore making PSR a valuable diagnostic tool in field conditions.     

Similar diagnostic performances of antigen detection and nucleic acid detection of Cryptosporidium spp. in a low-prevalence setting

The diagnostic value of a real-time PCR assay for the detection of Cryptosporidium spp. in fecal samples was assessed as compared to the combination of the immunocromatographic assay (IC) and immunofluorescence assay (IF) currently performed in our laboratory for the diagnosis of cryptosporidiosis. On a total of 1040 samples collected from 2006 to 2010 and belonging to 533 patients suspected of having an intestinal parasitosis, Cryptosporidium spp. was detected in 31 samples (belonging to 12 patients) by IC and IF; the real-time PCR assay revealed Cryptosporidium spp. DNA in 5 additional samples for a total of 36 samples (13 patients). The real-time PCR assay exhibited higher sensitivity than IC and IF; however, its application to the diagnosis of cryptosporidiosis should be evaluated by every single laboratory, depending on the availability of trained personnel, financial resources, and the cost/effectiveness related to the prevalence of cryptosporidiosis.