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11.
Sara A Bickersmith William Lainhart Marta Moreno Virginia M Chu Joseph M Vinetz Jan E Conn 《Memórias do Instituto Oswaldo Cruz》2015,110(4):573-576
We describe a simple method for detection of Plasmodium vivax and
Plasmodium falciparum infection in anophelines using a triplex
TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the
assay on Anopheles darlingi and Anopheles stephensi
colony mosquitoes fed with Plasmodium-infected blood meals and in
duplicate on field collected An. darlingi. We compared the real-time
PCR results of colony-infected and field collected An. darlingi,
separately, to a conventional PCR method. We determined that a cytochrome
b-PCR method was only 3.33% as sensitive and 93.38% as specific
as our real-time PCR assay with field-collected samples. We demonstrate that this
assay is sensitive, specific and reproducible. 相似文献
12.
Erin P. Price Derek S. Sarovich Elizabeth Nosworthy Jemima Beissbarth Robyn L. Marsh Janessa Pickering Lea-Ann S. Kirkham Anthony D. Keil Anne B. Chang Heidi C. Smith-Vaughan 《BMC genomics》2015,16(1)
Background
Haemophilus influenzae is an opportunistic bacterial pathogen that exclusively colonises humans and is associated with both acute and chronic disease. Despite its clinical significance, accurate identification of H. influenzae is a non-trivial endeavour. H. haemolyticus can be misidentified as H. influenzae from clinical specimens using selective culturing methods, reflecting both the shared environmental niche and phenotypic similarities of these species. On the molecular level, frequent genetic exchange amongst Haemophilus spp. has confounded accurate identification of H. influenzae, leading to both false-positive and false-negative results with existing speciation assays.Results
Whole-genome single-nucleotide polymorphism data from 246 closely related global Haemophilus isolates, including 107 Australian isolate genomes generated in this study, were used to construct a whole-genome phylogeny. Based on this phylogeny, H. influenzae could be differentiated from closely related species. Next, a H. influenzae-specific locus, fucP, was identified, and a novel TaqMan real-time PCR assay targeting fucP was designed. PCR specificity screening across a panel of clinically relevant species, coupled with in silico analysis of all species within the order Pasteurellales, demonstrated that the fucP assay was 100 % specific for H. influenzae; all other examined species failed to amplify.Conclusions
This study is the first of its kind to use large-scale comparative genomic analysis of Haemophilus spp. to accurately delineate H. influenzae and to identify a species-specific molecular signature for this species. The fucP assay outperforms existing H. influenzae targets, most of which were identified prior to the next-generation genomics era and thus lack validation across a large number of Haemophilus spp. We recommend use of the fucP assay in clinical and research laboratories for the most accurate detection and diagnosis of H. influenzae infection and colonisation.Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1857-x) contains supplementary material, which is available to authorized users. 相似文献13.
Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531 总被引:12,自引:0,他引:12
Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM)
cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative
and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed.
In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445
and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445
and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative
detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed,
which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits
of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were
10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter,
five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR
systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as
bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed
as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531.
The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established
event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for
GM cotton identification and quantification. 相似文献
14.
We investigated three probe design strategies used in quantitative polymerase chain reaction (PCR) for sensitivity in detection of the PCR amplicon. A plasmid with a 120-bp insert served as the DNA template. The probes were TaqMan, conventional molecular beacon (MB), and shared-stem molecular beacon (ATssMB and GCssMB). A shared-stem beacon probe combines the properties of a TaqMan probe and a conventional molecular beacon. It was found that the overall sensitivities for the four PCR probes are in the order of MB>ATssMB>GCssMB>TaqMan. The fluorescence quantum yield measurements indicate that incomplete or partial enzymatic cleavage catalyzed by Taq polymerase is the likely cause of the low sensitivities of two shared-stem beacons when compared with the conventional beacon probe. A high-fluorescence background associated with the current TaqMan probe sequence contributes to the relatively low detection sensitivity and signal-to-background ratio. The study points out that the nucleotide environment surrounding the reporting fluorophore can strongly affect the probe performance in real-time PCR. 相似文献
15.
CARITA M. ELFSTROM CHRISTIAN T. SMITH JAMES E. SEEB 《Molecular ecology resources》2006,6(4):1255-1259
We characterize 32 single nucleotide polymorphism genotyping assays for resolving genotypic variation in sockeye salmon Oncorhynchus nerka in the Pacific Rim. These assays are based on the 5′‐nuclease reaction and thus facilitate high‐throughput genotyping with minimal optimization time. Minor allele frequency differences (Δq) among collections were between 4.7% and 97.9%, resulting in per locus FST estimates of 0.02–0.71 with an average of 0.22. 相似文献
16.
Vibrio parahaemolyticus is recognized as a leading human food-borne pathogen. A TaqMan PCR assay based on the gyrase B gene (gyrB) sequence of V. parahaemolyticus was developed for quantitative detection of V. parahaemolyticus in seafood. The study involving 27 V. parahaemolyticus and 10 strains of other species indicated that the real-time PCR test was highly specific. The sensitivity of the assay was approximately a single CFU per PCR in pure culture and six to eight CFU per PCR in spiked raw oyster, respectively. Real-time PCR values of artificially inoculated oyster homogenates correlated well with plate counts determined using culture methods. A total of 300 seafood samples were analyzed and 78 (26%) of these samples were positive for V. parahaemolyticus using a conventional culture method and 97 (32.3%) using the real-time PCR assay. All culture-positive samples were PCR-positive. However, 19 samples positive by PCR were culture-negative. The results show that retail seafood is commonly contaminated with V. parahaemolyticus in harvest season in eastern China. These data also indicate that real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods. 相似文献
17.
Experiments were conducted on the ability of TaqMan molecular probes to detect plaice Pleuronectes platessa DNA from eggs, and cod Gadus morhua DNA from eggs and larvae following ingestion by a teleost predator, whiting Merlangius merlangus. Estimated half-life detection rate (T50) for eggs was 31 h, and 26 h for larvae, with some positive detections occurring even after visual inspection indicated complete gut clearance. Because TaqMan probes are taxon specific, the results presented demonstrate that this technique can provide a means of rapid and unambiguous detection of predation by teleosts on fish eggs and larvae. 相似文献
18.
19.
铜绿假单胞菌是临床上常见致病菌, 传统的检测方法有各种弊端。本研究对该细菌的ETA基因用生物信息学方法加以分析, 选取相对保守且高度特异的DNA序列, 设计一对特异性引物和一个TaqMan探针, 建立FQ-PCR (fluorescence quantitative PCR)检测PA的方法。通过对梯度浓度的铜绿假单胞菌基因组DNA样品进行FQ-PCR检测和对多种细菌的DNA进行扩增, 来检测其灵敏度和验证引物和探针的特异性。试验结果表明, 对比现有的检测方法, 以ETA基因为靶基因, 基于TaqMan探针的快速FQ-PCR检测技术有更高的灵敏度和更好的特异性等优点, 具有很好的研究价值和应用前景。 相似文献
20.
Kilic A Guclu AU Senses Z Bedir O Aydogan H Basustaoglu AC 《Antonie van Leeuwenhoek》2008,94(4):607-614
Methicillin-resistant Staphylococcus aureus (MRSA) cause serious community-acquired and nosocomial diseases all over the world. We determined the SCCmec types and occurrence of the PVL gene by using TaqMan real-time PCR method, and correlated these with phenotypic antibiotic
susceptibility patterns for MRSA strains collected from Gulhane Military Medical Academy Hospital (GMMAH) during 4 years study
period. To our knowledge, this is the first report from Turkey of molecular SCCmec typing analysis of MRSA stains. A total of 385 clinical MRSA isolates collected in the clinical and Microbiology Laboratory
at GMMAH between 2003 and 2006 were included in the study. Overall, SCCmec types-I, II, III, IV, V, nontypeable and PVL occurrence
were detected in 11 (2.8%), 3 (0.8%), 316 (82.1%), 20 (5.1%), 20 (5.1%), 15 (3.9%) and 5 (1.3%) isolates, respectively. A
total of 330 (85.5%) were SCCmec-I/II/III and 40 (10.3%) were SCCmec IV/V. SCCmec-I/II/III isolates were recovered more from
patients with serious infections in surgical departments especially those with intensive care units than the SCCmec-IV/V isolates (χ2 = 13.560, P < 0.001). SCCmec-I/II/III MRSA strains were predominantly recovered from blood stream (53.0%, P = 0.014), while SCCmec-IV/V strains were predominately isolated from skin and soft tissue and abscess (55.0%, P < 0.001). The PVL gene was detected in 10.0% of SCCmec-IV/V isolates in contrast to 0.3% in SCCmec-I/II/III (χ2 = 25.164, P < 0.001). SCCmec-I/II/III MRSA strains were more resistant to clindamycin (χ2 = 5.078, P = 0.024), amoxicillin-clavulanate (χ2 = 84.912, P < 0.001), erythromycin (χ2 = 4.651, P = 0.031), gentamicin (χ2 = 24.869, P < 0.001), and rifampin (χ2 = 18.878, P < 0.001) than SCCmec-IV/V MRSA strains. This data indicates that SCCmec-III MRSA strains that do not carry the PVL gene are the predominant MRSA
strains in our hospital setting in Ankara, capital of Turkey and that SCCmec-I/II/III MRSA strains may cause serious infections
in surgical departments especially those with intensive care units. 相似文献