应用TaqMan荧光定量PCR检测土拉弗朗西斯菌

目的：利用Roche LightCycler实时定量PCR系统建立一种快速、灵敏、特异的检测土拉弗朗西斯菌的方法。方法：基于TaqMan荧光探针实时定量PCR技术,选择土拉弗朗西斯菌染色体上的特异序列[醇醛酮还原酶（AKR）和外膜蛋白FopA基因]作为检测靶序列,建立土拉弗朗西斯菌实时定量PCR检测方法;评价该检测方法的特异性和灵敏性;采用克隆菌株污染环境土壤来模拟实际样品,评价该检测方法在快速检测与现场检测等实际应用中的表现。结果：优化筛选基因组中的FT-AKR和FT-fopA片段作为检测靶序列,所建立的土拉弗朗西斯菌实时定量PCR检测方法检测克隆菌株质粒的灵敏度均为10个拷贝/每个反应体系;以其他非土拉弗朗西斯菌为模板未出现非特异扩增;模拟环境土壤样品检测灵敏度2个引物对分别为440和960CFU/g土壤;盲测实验结果显示对于灵敏度范围内的阳性样本均能正确识别,并能正确检测出不同浓度的阳性样本。以FT-fopA片段为靶序列的扩增效率不及基于FT-AKR引物对的扩增。结论：基于FT-AKR片段的引物对扩增效率高,检测土拉弗朗西斯菌具有特异、灵敏的特点,对临床诊断、环境污染监测、防治生物突发事件等具有重要意义。     

A Multi-Target Real-Time PCR Assay for Rapid Identification of Meningitis-Associated Microorganisms

A central nervous system (CNS) infection, such as meningitis, is a serious and life-threatening condition. Bacterial meningitis can be severe and may result in brain damage, disability or even death. Rapid diagnosis of CNS infections and identification of the pathogenic microorganisms are needed to improve the patient outcome. Bacterial culture of a patient??s cerebrospinal fluid (CSF) is currently considered the ??gold standard?? for diagnosing bacterial meningitis. From the CSF cultures researchers can assess the in vitro susceptibility of the causative microorganism to determine the best antibiotic treatment. However, many of the culture assays, such as microscopy and the latex agglutination test are not sensitive. To enhance pathogen detection in CSF samples we developed a multi-target real-time PCR assay that can rapidly identify six different microorganisms: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes and Cryptococcus neoformans. In this study we applied this PCR analysis to 296 CSF samples from patients who were suspected of having meningitis. Of the 296 samples that were examined, 59 samples were positive according to the CSF culture and/or molecular assays. Forty-six CSF samples were positive for both the CSF culture and our real-time PCR assay, while 13 samples were positive for the real-time PCR but negative for the traditional assays. This discrepancy may have been caused by the fact that these samples were collected from 23 patients who were treated with antimicrobials before CSF sampling.     

Identification of Francisella tularensis in natural water samples by PCR

Abstract Francisella tularensis , the causative agent of the epizootic disease tularemia in mammals, can be isolated from mud and water. To study the spread and persistence of Francisella tularensis in water, different strategies for pre-treatment of natural water samples prior to identification of the bacterium by polymerase chain reaction (PCR) were evaluated. A method for handling of samples taken from natural waters was developed. Applied on natural water samples amended with F. tularensis , the method rendered identification by PCR reproducible and it resulted in an amplified Francisella -specific product in all samples from natural waters tested. In addition, by employing primers targeting conserved regions of the 16S rDNA the presence of bacteria was demonstrated in all samples investigated. The results presented will, in combination with other techniques that allow identification, improve studies on the epizootiology and epidemiology of the genus Francisella .     

TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify Francisella tularensis and Its Subspecies and Subpopulations

Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.     

Development of a Real-Time PCR for Identification of Brachyspira Species in Human Colonic Biopsies

Background

Brachyspira species are fastidious anaerobic microorganisms, that infect the colon of various animals. The genus contains both important pathogens of livestock as well as commensals. Two species are known to infect humans: B. aalborgi and B. pilosicoli. There is some evidence suggesting that the veterinary pathogenic B. pilosicoli is a potential zoonotic agent, however, since diagnosis in humans is based on histopathology of colon biopsies, species identification is not routinely performed in human materials.

Methods

The study population comprised 57 patients with microscopic evidence of Brachyspira infection and 26 patients with no histopathological evidence of Brachyspira infection. Concomitant faecal samples were available from three infected patients. Based on publically available 16S rDNA gene sequences of all Brachyspira species, species-specific primer sets were designed. DNA was extracted and tested by real-time PCR and 16S rDNA was sequenced.

Results

Sensitivity and specificity for identification of Brachyspira species in colon biopsies was 100% and 87.7% respectively. Sequencing revealed B. pilosicoli in 15.4% of patients, B. aalborgi in 76.9% and a third species, tentatively named “Brachyspira hominis”, in 26.2%. Ten patients (12.3%) had a double and two (3.1%) a triple infection. The presence of Brachyspira pilosicoli was significantly associated with inflammatory changes in the colon-biopsy (p = 0.028).

Conclusions

This newly designed PCR allows for sub-differentiation of Brachyspira species in patient material and thus allows large-scaled surveillance studies to elucidate the pathogenicity of human Brachyspira infections. One-third of affected patients appeared to be infected with a novel species.     

TaqMan实时荧光定量PCR鉴定燕窝方法的建立

目的:燕窝是一种名贵药材,但目前市场上有很多假冒产品,因此需要建立一种简便可靠的鉴定方法。方法:通过荧光定量PCR对燕窝样品进行检测,对雨燕属、金丝燕属和其他物种细胞色素b基因的序列进行分析。结果:设计了一条特异靶向雨燕属和金丝燕属细胞色素b基因的TaqMan探针,发现此探针具有良好的特异性和灵敏度,可检测痕量的燕窝样品,对其他物种的检测结果为阴性。结论:所设计的TaqMan探针和实时荧光PCR方法可应用于燕窝的真伪鉴别,准确性高、实用性强。     

Comparison of PCR-RFLP with 21-plex PCR and rDNA Sequencing for Identification of Clinical Yeast Isolates

Non-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which are time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR. One hundred and seventy-three yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method. The results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=?C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR-RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR. The application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

     

Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.     

Multi-probe Real-Time PCR Identification of Four Common Candida Species in Blood Culture Broth

We developed a single-tube real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting Candida albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Primers were designed to amplify 18S rRNA gene of the genus Candida, and DNA probes were designed to hybridize two areas of the amplicons. The amplification curves and specific melting peaks of the probes hybridized with PCR product were used for definite species identifications. The reaction specificity was 100 % when evaluating the assay using DNA samples from 21 isolates of fungal and bacterial species. The assay was further evaluated in 129 fungal blood culture broth samples which were culture positive for fungus. Of the 129 samples, 119 were positively identified as: C. albicans (39), C. tropicalis (30), C. parapsilosis (23), C. glabrata (20), Candida spp. (5), and two samples containing mixed C. glabrata/C. albicans and C. glabrata/C. tropicalis. The five Candida spp. were identified by sequencing analysis as C. krusei, C. dubliniensis, C. aquaetextoris, and two isolates of C. athensensis. Of the ten samples which showed negative PCR results, six were Cryptococcus neoformans, and the others were Trichosporon sp., Rhodotorula sp., Fusarium sp., and Penicillium marneffei. Our findings show that the assay was highly effective in identifying the four medically important Candida species. The results can be available within 3 h after positivity of a blood culture broth sample.     

Real-Time PCR and Sequencing Assays for Rapid Detection and Identification of Avian Schistosomes in Environmental Samples

Cercarial dermatitis, also known as swimmer''s itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed. A PCR assay was also developed to amplify a 28S rDNA region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded with Schistosoma mansoni cercariae, the 18S rDNA TaqMan assay enabled detection at a level of 5 S. mansoni cercariae in 100 liters of lake water. The 18S rDNA TaqMan and 28S rDNA PCR sequencing assays were also applied to 100-liter water samples collected from lakes in Nebraska and Wisconsin where there were reported dermatitis outbreaks. Avian schistosome DNA was detected in 11 of 34 lake water samples using the TaqMan assay. Further 28S rDNA sequence analysis of positive samples confirmed the presence of avian schistosome DNA and provided a preliminary identification of the avian schistosomes in 10 of the 11 samples. These data indicate that the broadly schistosome-reactive TaqMan assay can be effective for rapid screening of large-volume water samples for detection of avian schistosomes, thereby facilitating timely response actions to mitigate or prevent dermatitis outbreaks. Additionally, samples positive by the 18S rDNA TaqMan assay can be further assayed using the 28S rDNA sequencing assay to both confirm the presence of schistosomes and contribute to their identification.     

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S.cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 × 10⁵ to 3.8 CFU/ml in sweet wine and from 5 × 10⁶ to 50CFU/ml in red wine.     

Identification and Characterization of Pathogenic Yersinia enterocolitica Isolates by PCR and Pulsed-Field Gel Electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n = 48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

A Targeted Real-Time PCR Assay for Studying Naphthalene Degradation in the Environment

A quantitative real-time polymerase chain reaction (PCR) assay was developed for monitoring naphthalene degradation during bioremediation processes. The phylogenetic affiliations of known naphthalene-hydroxylating dioxygenase genes were determined to target functionally related bacteria, and degenerate primers were designed on the basis of the close relationships among dioxygenase genes identified from naphthalene-degrading Proteobacteria. Evaluation of the amplification specificity demonstrated that the developed real-time PCR assay represents a rapid, precise means for the group-specific enumeration of naphthalene-degrading bacteria. According to validation with bacterial pure cultures, the assay discriminated between the targeted group of naphthalene dioxygenase sequences and genes in other naphthalene or aromatic hydrocarbon-degrading bacterial strains. Specific amplification of gene fragments sharing a high sequence similarity with the genes included in the assay design was also observed in soil samples recovered from large-scale remediation processes. The target genes could be quantified reproducibly at over five orders of magnitude down to 3 × 10² gene copies. To investigate the suitability of the assay in monitoring naphthalene biodegradation, the assay was applied in enumerating the naphthalene dioxygenase genes in a soil slurry microcosm. The results were in good agreement with contaminant mineralization and dot blot quantification of nahAc gene copies. Furthermore, the real-time PCR assay was found to be more sensitive than hybridization-based analysis.     

Identification and Validation of Suitable Housekeeping Genes for Normalizing Quantitative Real-Time PCR Assays in Injured Peripheral Nerves

Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.     

Diagnostic Real-Time PCR for Detection of Salmonella in Food

A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10³ CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10⁴ CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.     

A New Real-Time PCR for the Detection of Plasmodium ovale wallikeri

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite’s small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 11 to P. ovale wallikeri. Thus, this assay provides a useful tool that can be applied to epidemiological investigations of the two newly recognized distinct P. ovale species, that might reveal if these species also differ in their clinical manifestation, drugs susceptibility and relapse periodicity. The results presented confirm that P. ovale wallikeri is not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired their P. ovale infection in African countries, mostly situated in West Africa.