Evaluation of new gyrB-based real-time PCR system for the detection of B. fragilis as an indicator of human-specific fecal contamination

A rapid and specific gyrB-based real-time PCR system has been developed for detecting Bacteroides fragilis as a human-specific marker of fecal contamination. Its specificity and sensitivity was evaluated by comparison with other 16S rRNA gene-based primers using closely related Bacteroides and Prevotella. Many studies have used 16S rRNA gene-based method targeting Bacteroides because this genus is relatively abundant in human feces and is useful for microbial source tracking. However, 16S rRNA gene-based primers are evolutionarily too conserved among taxa to discriminate between human-specific species of Bacteroides and other closely related genera, such as Prevotella. Recently, one of the housekeeping genes, gyrB, has been used as an alternative target in multilocus sequence analysis (MLSA) to provide greater phylogenetic resolution. In this study, a new B. fragilis-specific primer set (Bf904F/Bf958R) was designed by alignments of 322 gyrB genes and was compared with the performance of the 16S rRNA gene-based primers in the presence of B. fragilis, Bacteroides ovatus and Prevotella melaninogenica. Amplicons were sequenced and a phylogenetic tree was constructed to confirm the specificity of the primers to B. fragilis. The gyrB-based primers successfully discriminated B. fragilis from B. ovatus and P. melaninogenica. Real-time PCR results showed that the gyrB primer set had a comparable sensitivity in the detection of B. fragilis when compared with the 16S rRNA primer set. The host-specificity of our gyrB-based primer set was validated with human, pig, cow, and dog fecal samples. The gyrB primer system had superior human-specificity. The gyrB-based system can rapidly detect human-specific fecal source and can be used for improved source tracking of human contamination.     

Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae

Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.     

山羊痘病毒TaqMan-MGB荧光定量PCR检测方法的建立与应用

利用DNAStar分析了GenBank中所有8株羊痘病毒全基因序列,选取位于山羊痘病毒(AY077835)基因组gp064区域约64bp的基因片段,设计并合成了一对PCR引物和一条TaqMan-MGB探针,建立了FQ-PCR和标准曲线,并利用该方法对山羊痘临床皮肤痘疹材料和人工感染动物材料进行GPV核酸检测。结果表明,构建的FQ-PCR具有良好的敏感性、特异性、稳定性和临床应用性。该方法的建立为山羊痘临床快速高效地诊断和山羊痘病毒感染羊只的发生、发展及转归研究提供了一种有效的手段。     

A duplex real-time PCR assay for detection of mutT4 and mutT2 mutations in Mycobacterium tuberculosis of Beijing genotype

To determine the polymorphism of mutT genes of Mycobacterium tuberculosis of Beijing genotype, we developed a duplex real-time PCR assay based on hybridization probes for the Roche LightCycler instrument. The assay rapidly detects mutations at codons 48 and 58 of genes mutT4 and at mutT2, respectively.     

A real-time PCR diagnostic method for detection of Naegleria fowleri

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence.Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.     

Development of a real-time PCR assay for detection of Mytilus species specific alleles: Application to a sampling survey in Scotland

Shellfish aquaculture is a growing industry in Scotland, dominated by the production of the mussel Mytilus edulis, the native species. Recently the discovery of Mytilus galloprovincialis and Mytilus trossulus together with M. edulis and all 3 hybrids in cultivation in some Scottish sea lochs led to questions regarding the distribution of mussel species in Scotland. The establishment of an extensive sampling survey, involving the collection of mussels at 34 intertidal sites and 10 marinas around Scotland, motivated the development of a high-throughput method for identification of Mytilus alleles from samples. Three Taqman®-MGB probes and one set of primers were designed, based on the previously described Me 15/16 primers targeting the adhesive protein gene sequence, and samples were screened for the presence of M. edulis, M. galloprovincialis and M. trossulus alleles using real-time PCR. Mytilus edulis alleles were identified in samples from all 44 sites. Mytilus galloprovincialis alleles were found together with M. edulis alleles extensively in northern parts of the west and east coasts. Mytilus trossulus alleles were identified in samples from 6 sites in the west and south-west of Scotland. Because M. trossulus is generally undesirable in cultivation and therefore preventing the geographical spread of this species across Scotland is considered beneficial by the shellfish aquaculture industry, these 6 samples were further analysed for genotype frequencies using conventional PCR. Although distribution of the non-native species M. galloprovincialis and M. trossulus have proven to be more widespread than previously thought, there is no evidence from our study of either M. trossulus or M. galloprovincialis acting as an invasive species in Scotland. The real-time PCR method developed in this study has proven to be a rapid and effective tool for the identification of M. edulis, M. galloprovincialis and M. trossulus alleles from samples and should prove useful in future surveys, ecological or aquaculture management related studies in both unispecific and mixed species areas of these species.     

Optimal swab processing recovery method for detection of bioterrorism-related Francisella tularensis by real-time PCR

Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5′nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations.     

Evaluation of real-time PCR targeting hbb gene for Borrelia species identification

Several molecular methods have been employed for Borrelia species identification. Newly developed technology, real-time polymerase chain reaction (RT-PCR), combines simultaneous amplification, detection and differentiation of strains in one PCR run. The aim of the study was to perform and evaluate RT-PCR for Borreliaburgdorferi sensu lato species identification. Borrelia species identification was accomplished on 374 Borrelia strains using two approaches: 1.) MluI restriction of entire borrelial chromosome (MluI-large restriction fragment patterns, LRFP), and 2.) RT-PCR targeting hbb gene and specific melting temperature (Tm) detection. The results of the two molecular methods were compared. With MluI-RFLP we were able to differentiate all Borrelia species and their subtypes within particular species. RT-PCR based on Tm determination identified unique strains within the species Borreliaafzelii (Tm 66.11 °C), B. burgdorferi sensu stricto (Tm 68.18 °C), Borreliaspielmanii (Tm 59.45 °C) and Borreliavalaisiana (Tm 59.62 °C). We were not able to distinguish the last two species that shared almost identical Tm. The large majority of Borreliagarinii strains shared Tm 51.42 °C, while subtype Mlg4 was characterized by Tm 56.87 °C. Strains of Borrelialusitaniae species also were heterogeneous; human isolate had Tm 63.47 °C while two tick isolates shared Tm 61.77 °C. Differences inside hbb gene enabled differentiation of the majority of Borrelia species, and revealed two clusters within B. garinii and B. lusitaniae species, respectively, but it was not possible to distinguish B. spielmanii form B. valaisiana. The major advantage of RT-PCR was that it was easy to perform and that the results were obtained within a few hours.     

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

Introduction

Acquired AmpC enzymes, classified as miscellaneous extended-spectrum β-lactamase (ESBL_M) enzymes according to a recently proposed β-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL_M are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla_AmpC detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes.

Material and methods

Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/− cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla_AmpC real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing.

Results and discussion

The real-time PCR assay was able to detect and sub-classify all acquired bla_AmpC genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla_AmpC real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.     

Combining ethidium monoazide treatment with real-time PCR selectively quantifies viable Batrachochytrium dendrobatidis cells

Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10^® Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.     

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1 pg of purified genomic DNA, 5 EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent.     

Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay

Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking.     

Quantitative detection of probiotic Bifidobacterium strains in bacterial mixtures by using real-time PCR

Strain-specific rRNA-targeted primers were designed for the quantitative detection of Bifidobacterium infantis Y1, B. breve Y8 and B. longum Y10 used in a pharmaceutical probiotic product (VSL-3). PCR and real-time PCR techniques with the selected primers were employed for the direct enumeration of the bifidobacteria in the probiotic preparation and for studying their kinetic characteristics in batch cultures. These analysis revealed that B. infantis Y1 was the predominant strain in the probiotic product and that its growth rate was the highest. Since B. infantis Y1, B. breve Y8 and B. longum Y10 are co-cultured during the industrial production of VSL-3, the kinetic characteristics of these strains can explain their different concentrations in the probiotic preparation. A validation of the PCR quantification method was performed by identifying a representative number of isolates from the bacterial mixtures with automated ribotyping. The methodology described represents a useful tool for the specific quantitative detection of bacterial strains and species in complex mixtures such as pharmaceutical preparations, dairy starter cultures, faecal samples and biopsies.     

Quadruplex real-time quantitative PCR assay for the detection of pathogens related to late-onset ventilator-associated pneumonia: A preliminary report

A quadruplex real-time (RT) qPCR assay for the detection and quantification in 4 h of Staphylococcusaureus, Pseudomonasaeruginosa, Acinetobacterbaumannii and Stenotrophomonasmaltophilia directly from bronchoalveolar lavage specimens was developed. The specificity of the assay was 100% for all four species.     

A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector

Transmission-blocking vaccines prevent the development of Plasmodium parasite within the mosquito vector, thereby thwarting the spread of malaria through a community. The gold standard for determining the efficacy of a transmission-blocking vaccine is the standard membrane feeding assay. This assay requires the dissection of mosquitoes and microscopic counting of oocysts present on the mosquito mid-gut, typically at 7-10 days p.i. Here we describe a real-time quantitative PCR assay that is rapid, target-specific and robust, with a sensitive detection threshold and which may be employed earlier p.i. than the standard membrane feeding assay and is applicable to preserved material. The real-time PCR assay utilises the LightCycler platform and SYBR Green I detection system to amplify 180 bp of the asexual form of the Plasmodium falciparum rRNA gene. It has a quantitative range of greater than four orders of magnitude and a detection threshold of 10 parasites. Validation experiments using a monoclonal antibody of known blocking activity revealed the real-time PCR assay to give equivalent results to the standard membrane feeding assay. In addition, the PCR assay can establish the effect of such a monoclonal antibody on the parasites' development within the oocyst and on the sporozoite (the transmissible stage) yield, providing a more pertinent assessment of transmission blocking activity than is possible by the standard membrane feeding assay. This assay may also be employed to monitor the sporogonic development of P. falciparum parasites within the mosquito vector.     

Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee

The incidence of nosemosis has increased in recent years due to an emerging infestation of Nosema ceranae in managed honey bee populations in much of the world. A real-time PCR assay was developed to facilitate detection and quantification of both Nosema apis and N. ceranae in both single bee and pooled samples. The assay is a multiplexed reaction in which both species are detected and quantified in a single reaction. The assay is highly sensitive and can detect single copies of the target sequence. Real-time PCR results were calibrated to spore counts generated by standard microscopy procedures. The assay was used to assess bees from commercial apiaries sampled in November 2008 and March 2009. Bees from each colony were pooled. A large amount of variation among colonies was evident, signifying the need to examine large numbers of colonies. Due to sampling constraints, a subset of colonies (from five apiaries) was sampled in both seasons. In November, N. apis levels were 1212 ± 148 spores/bee and N. ceranae levels were 51,073 ± 31,155 spores/bee. In March, no N. apis was detected, N. ceranae levels were 11,824 ± 6304 spores/bee. Changes in N. ceranae levels were evident among apiaries, some increasing and other decreasing. This demonstrates the need for thorough sampling of apiaries and the need for a rapid test for both detection and quantification of both Nosema spp. This assay provides the opportunity for detailed study of disease resistance, infection kinetics, and improvement of disease management practices for honey bees.     

Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ mutant vaccine strain used for DIVA-strategies

Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ⁻ mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ⁻ mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ⁻ mutant vaccine strain.     

Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes

Listeriosis is a serious food-borne infection with mortality rates approaching 30%. Therefore, the rapid, cost-effective, and automated detection of Listeria monocytogenes throughout the food chain continues to be a major concern. Here we describe three novel quantitative real-time PCR assays for L. monocytogenes based on amplification of a target hlyA gene with SYBR Green I chemistry and hydrolysis probe (TaqMan MGB probe). In order to offer sensitive, rapid and robust tool of additional economical value the real-time PCR assays were designed and optimized to only 5 μl-reactions. All assays were evaluated by using different non-reference Listeria strains isolated from various food matrices. Results demonstrated specificity to L. monocytogenes with accurate quantification over a dynamic range of 5-6 log units with R² higher than 0.98 and amplification efficiencies reaching above 92%. The detection and quantification limits were as low as 165 genome equivalents. Comparison of novel assays to commercially available TaqMan® Listeria monocytogenes Detection Kit and previously published studies revealed similar specificity, sensitivity and efficiency, but greater robustness and especially cost-efficiency in the view of smaller reaction volumes and continuous increase in sample throughput.     

Differential diagnosis of Taenia asiatica using multiplex PCR

Taenia asiatica and T. saginata are frequently confused tapeworms due to their morphological similarities and sympatric distribution in Asian regions. To resolve this problem, a high-resolution multiplex PCR assay was developed to distinguish T. asiatica infections from infection with other human Taenia tapeworms. For molecular characterization, the species specificity of all materials used was confirmed by sequencing of the cox1 gene. Fifty-two samples were analyzed in this study, comprising 20 samples of T. asiatica genomic DNA from China, Korea, and the Philippines; 24 samples of T. saginata from Belgium, Chile, China, Ethiopia, France, Indonesia, Korea, Laos, the Philippines, Poland, Taiwan, Thailand, and Switzerland; and 10 samples of T. solium from Cape Verde, China, Honduras, and Korea. The diagnostic quality of the results obtained using PCR and species-specific primers designed from valine tRNA and NADH genes was equal to that based on the nucleotide sequencing of the cox1 gene. Using oligonucleotide primers Ta4978F, Ts5058F, Tso7421F, and Rev7915, the multiplex PCR assay was useful for the differentially diagnosing T. asiatica, T. saginata, and T. solium based on 706-, 629-, and 474-bp bands.     

Schistosoma japonicum: A PCR assay for the early detection and evaluation of treatment in a rabbit model

A specific PCR assay for the detection of Schistosoma japonicum DNA in rabbit fecal and serum samples was developed by amplifying a 230-bp fragment from the sequence information of the clone G55A of the highly repetitive retrotransposon SjR2. The minimum amount of DNA detectable using the PCR assay was 0.8 pg, and the expected PCR product was amplified when DNA equivalent of 1.1 egg from feces was used as template. In the meantime, serum anti-worm IgG was examined by ELISA. ELISA gave positive results at 4-6 weeks post-infection depending on the cercarial doses. The parasite eggs were detected in feces at 7 weeks post-infection. In contrast, S. japonicum DNA was detected in sera at first week post-infection, and it became negative at 10 weeks post-treatment, whereas the anti-worm IgG was still at high levels at 23 weeks post-treatment. These data demonstrated that the PCR assay established provides a potential tool for the early diagnosis and therapy evaluation for S. japonicum infection in humans.