Multiplex PCR reveals that viruses are more frequent than bacteria in children with cystic fibrosis

BackgroundCystic fibrosis is a degenerative disease characterized by progressive epithelial secretory gland dysfunction associated with repeated respiratory infections. Bacterial infections are very frequent in children with cystic fibrosis, but because rapidMethodsfor screening for the wide variety of potentially involved viruses were unavailable until recently, the frequency of viral presence is unknown. Multiplex PCR enables screening for many viruses involved in respiratory infections.ObjectivesThis study aimed to evaluate the frequency of viruses and bacteria in respiratory specimens from children with cystic fibrosis and to clarify the incidence and characteristics (seasonality and age of patients) of different viruses detected in children with cystic fibrosis.Study designIn this 2-year prospective study, we obtained paired nasopharyngeal-swab and sputum specimens from children with cystic fibrosis during clinical respiratory examinations separated by at least 14 days. We analyzed viruses in nasopharyngeal-swab specimens with multiplex PCR and bacteria in sputum with standard methods.ResultsWe analyzed 368 paired specimens from 33 children. We detected viruses in 154 (41.8%) and bacteria in 132 (35.9%). Bacteria were commoner in spring and summer; viruses were commoner in autumn and winter. In every season, Staphylococcus aureus was the commonest bacteria and rhinovirus was the commonest virus. Nearly all infections with Haemophilus influenzae occurred in autumn and winter.Viruses were more prevalent in children <5 years old, and bacteria were more prevalent in children ≥12 years old.ConclusionsMultiplex PCR screening for respiratory viruses is feasible in children with cystic fibrosis; the clinical implications of screening warrant further study.     

Triplex PCR‐based detection of enterotoxigenic Bacillus cereus ATCC 14579 in nonfat dry milk

Although many strains of Bacillaceae are considered nonpathogenic, Bacillus cereus is recognized worldwide as a bacterial pathogen in a variety of foods. The ability of B. cereus to cause gastroenteritis following ingestion of contaminated food is due to the production of enterotoxins. The ubiquity of this genus makes it a persistent problem for quality assurance in food processing environments. The primary objective of this study was to develop and apply a multiplex real‐time PCR‐based assay for rapid and sensitive detection of enterotoxigenic B. cereus. Template DNA was separately extracted from tryptic soy broth (TSB)‐grown and 2.5% Nonfat Dry Milk (NFDM)‐grown B. cereus using a commercial system. Three enterotoxin gene fragments (hblC, nheA, and hblA) were simultaneously amplified in real‐time followed by melting curve analysis to confirm amplicon identity. Resolution of melting curves (characteristic T_m) was achieved for each amplicon (hblC = 74.5 °C; nheA = 78 °C; and hblA = 85.5 °C in TSB and 84 °C in NFDM) with an assay sensitivities of 10¹ CFU/ml for both TSB and NFDM‐grown B. cereus compared to 10⁴ CFU/ml in either matrix using gel electrophoresis. The results demonstrate the potential sensitivity of real‐time bacterial detection methods in a heterogenous food matrix using real‐time PCR. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

多重PCR检测急性非淋巴细胞白血病IgH及TCRVγΙ-Jγ基因重排

目的:为进一步了解急性非淋巴细胞白血病(ANLL)病人基因重排情况。方法:应用多重PCR技术检测41例ANLL病人IgH及TCRVγIJγ基因重排。结果:26.8%(11/41)ANLL病人存在IgH或/和TCRVγIJγ基因重排,3例病人同时存在IgH和TCRVγIJγ基因重排;基因重排阳性ANLL治疗缓解率低于重排阴性ANLL病人(P<0.05)。结论:①IgH或TCR基因重排并非局限于淋巴细胞白血病,也可发生于部分ANLL病人;②多重PCR技术可以一次PCR扩增同时检测两种重排基因,更适于临床推广应用。     

Multiplex high resolution melting assay for estimation of Porcine Endogenous Retrovirus (PERV) relative gene dosage in pigs and detection of PERV infection in xenograft recipients

Porcine Endogenous Retrovirus (PERV) poses an infectious risk in the field of xenotransplantation. This risk may be mitigated by breeding selectively animals bearing favorable PERV genetic characteristics including pigs with low levels of PERV integrated in the genome. A real-time quantitative polymerase chain reaction (PCR) assay employing the Roche High Resolution Melting (HRM) Master was used to estimate the relative gene dosage of PERV pol integrated within the pig genome. When assessed across 99 pigs of the Auckland Island breed numerous animals bearing low gene dosage were identified. The assay was adapted further to perform multiplex PCR for the detection of PERV infection within xenograft recipients. Besides PERV, amplification targets for the multiplex PCR include a pig cell marker for the determination of microchimerism and an internal amplification control (IAC) to assess the efficiency of nucleic acid isolation and effects of PCR inhibition. When 12 patients who had received porcine islet transplants were tested no evidence of PERV infection was found. The assay was shown to be specific, highly reproducible with superior performance over conventional nested PCR. This assay can be used as both a screening tool for PERV proviral levels within donor pigs and as a diagnostic tool to examine PERV transmission in human patients treated with porcine xenotransplantation material.     

Validation and comparison of two multiplex technologies, Luminex and Mesoscale Discovery, for human cytokine profiling

Biomarker research has rapidly expanded over recent years aided by the progressive development of research tools, in particular the different multiplex technologies allowing simultaneous measurement of multiple analytes. It is foreseeable that such technology will have an integral role in clinical studies for establishing biomarker profiles of disease status, but validation of the tools is essential to confirm the reliability of their application. More comparable studies between multiplex platforms are required to enable users to determine which of these are best for a particular clinical study, as different platforms will have varying levels of performance for the validation parameters. Comparison of two multiplex platforms, the Luminex^® and the Mesoscale Discovery, has been performed to determine their performance for the validation parameters of sensitivity, precision and accuracy for the cytokines IL-2, IL-4, IL-8, IL-10, IL-12, IFNγ and TNFα. When measuring high concentrations both platforms show good accuracy (within +/− 25% recovery) with all cytokines except IL-12 for the MSD. At low concentrations, +/− 25% recovery was seen with all cytokines except IL-2 and IL-8 for the Luminex and IL-2 and IL-12 for the MSD. Although quantitative differences are found, relative differences are comparable, and consequently both platforms have been shown to be suitable for analyzing trends in multiple cytokine profiles, with the Luminex having better precision and the Mesoscale Discovery having greater sensitivity.     

登革病毒悬液芯片分型检测方法的建立

目的 建立一种基于悬液芯片的登革病毒(dengue virus,DV)检测方法,可对四种血清型登革病毒进行快速检测和鉴定.方法 依据GenBank上4种病毒的基因序列信息,设计并合成相关引物及探针序列.抽提病毒RNA,经反转录后对目的基因进行PCR扩增,产物与核酸探针微球组杂交后于Bio-PlexTM 200系统检测荧光信号值.结果 DV1的悬液芯片检测敏感性约9 DNA拷贝,DV2、DV3、DV4的悬液芯片检测敏感性约90 DNA拷贝.进而将本方法用于检测15份临床标本,其检测结果与分型荧光RT-PCR一致.结论 建立了可同时检测四种血清型登革病毒的悬液芯片检测方法,为快速筛查和鉴定登革病毒提供了新的手段.     

A rapid and easy method for multiple endocrine neoplasia type 1 mutation detection using conformation-sensitive gel electrophoresis

Until now, the study of the multiple endocrine neoplasia type 1 (MEN1) gene in patients suspected of having the disease was expensive and laborious due to the large size of the gene. We have optimized the conformation-sensitive gel electrophoresis (CSGE) technique to analyze by four rather simple multiplex PCR reactions, and a single electrophoresis run, the entire coding region of the MEN1 gene, plus the exon–intron boundaries. This improvement of the CSGE technique was confirmed as an effective procedure for screening for the MEN1 gene by detecting ten previously known MEN1 gene mutations and four polymorphisms. The MEN1 gene of 12 patients with unknown mutations was then screened, and an abnormal CSGE profile was identified in 10/12 cases. Subsequent DNA sequencing demonstrated 3 of them to be novel mutations (E45K, 4479delACAG, 6073insC) and 7 to have been previously reported; in the remaining 2 patients, we confirmed the absence of any alteration of the coding sequence of MEN1. Mutation screening of the MEN1 gene using CSGE was demonstrated to be a fast, simple, and inexpensive method to study patients suspected of having MEN1 disease. Received: November 29, 2001 / Accepted: January 28, 2002     

Evaluation of the Roche LightMix Gastro parasites multiplex PCR assay detecting Giardia duodenalis,Entamoeba histolytica,cryptosporidia, Dientamoeba fragilis,and Blastocystis hominis

ObjectivesMultiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Blastocystis hominis, and Dientamoeba fragilis with routine laboratory procedures.MethodsStool specimens (n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica/dispar, and microscopy of wet mounts. Discrepant results were analysed by in-house PCR.ResultsD. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p < 0.0001) of 909 samples, respectively. Of 918 samples analysed for Cryptosporidium spp., six were positive by LMAGP (three could be confirmed by Kinyoun staining and one by in-house PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar. Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar. One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs (n = 3) and apathogenic amoebae (n = 27).ConclusionsThe data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics.     

三种呼吸道病毒实验室检测方法比较

目的在3种呼吸道多病原检测方法中筛选出敏感的实验室检测方法。方法以73份上呼道感染病例咽拭子标本作为检测对象,利用3种呼吸道多病原病毒检测方法,对17种呼吸道病毒检测指标进行平行检测,根据不同呼吸道病毒检测指标检出率,评价3种试剂检测效果。结果自建RT-PCR及PCR检测方法共检出56个阳性指标,市售多重RT-PCR检测试剂盒共检出41个阳性指标,市售多重实时荧光RT-PCR检测试剂盒共检出87个阳性指标。结论多重实时荧光RT-PCR检测试剂盒具有更高的检出率,可用于呼吸道多病原病毒的实验室检测。