scholarly journals A quadruplex real-time PCR assay for the detection of Yersinia pestis and its plasmids

2008 ◽  
Vol 57 (3) ◽  
pp. 324-331 ◽  
Author(s):  
Alvin Stewart ◽  
Benjamin Satterfield ◽  
Marissa Cohen ◽  
Kim O'Neill ◽  
Richard Robison
Keyword(s):  
Yersinia Pestis ◽  
Real Time ◽  
Real Time Pcr ◽  
Yersinia Pseudotuberculosis ◽  
Target Sequence ◽  
Health Departments ◽  
Pcr Assay ◽  
Virulence Plasmids ◽  
Taqman Probes ◽  
Sporadic Disease

Yersinia pestis, the aetiological agent of the plague, causes sporadic disease in endemic areas of the world and is classified as a National Institute of Allergy and Infectious Diseases Category A Priority Pathogen because of its potential to be used as a bioweapon. Health departments, hospitals and government agencies need the ability to rapidly identify and characterize cultured isolates of this bacterium. Assays have been developed to perform this function; however, they are limited in their ability to distinguish Y. pestis from Yersinia pseudotuberculosis. This report describes the creation of a real-time PCR assay using Taqman probes that exclusively identifies Y. pestis using a unique target sequence of the yihN gene on the chromosome. As with other Y. pestis PCR assays, three major genes located on each of the three virulence plasmids were included: lcrV on pCD1, caf1 on pMT1 and pla on pPCP1. The quadruplex assay was validated on a collection of 192 Y. pestis isolates and 52 near-neighbour isolates. It was discovered that only 72 % of natural plague isolates from the states of New Mexico and Utah harboured all three virulence plasmids. This quadruplex assay proved to be 100 % successful in differentiating Y. pestis from all near neighbours tested and was able to reveal which of the three virulence plasmids a particular isolate possessed.

scholarly journals Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

2015 ◽  
Vol 9 (5) ◽  
pp. e0003765 ◽  
Author(s):  
Carolina I. Cura ◽  
Tomas Duffy ◽  
Raúl H. Lucero ◽  
Margarita Bisio ◽  
Julie Péneau ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Taqman Probes

PCR and culture for diagnosis of Acanthamoeba keratitis

2020 ◽  
pp. bjophthalmol-2020-316730
Author(s):  
Helene Yera ◽  
Vichita Ok ◽  
Fiona Lee Koy Kuet ◽  
Naima Dahane ◽  
Frédéric Ariey ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Latent Class ◽  
Rrna Gene ◽  
Target Sequence ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Corneal Scraping ◽  
Pcr Assays

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

scholarly journals Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis

2005 ◽  
Vol 51 (10) ◽  
pp. 1778-1785 ◽  
Author(s):  
Catherine J Chase ◽  
Melanie P Ulrich ◽  
Leonard P Wasieloski ◽  
John P Kondig ◽  
Jeffrey Garrison ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Real Time ◽  
Real Time Pcr ◽  
Yersinia Pseudotuberculosis ◽  
Insertion Site ◽  
Closely Related Species ◽  
Yersinia Species ◽  
Pcr Assays ◽  
Probe Assay ◽  
Selective Differentiation

Abstract Background: Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results. Methods: Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan® minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe® and MGB Eclipse™ probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis. Results: The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 °C Tm difference and the MGB Eclipse probe a slightly more than 4 °C difference. Conclusions: Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.

A novel single-tube multiplex real-time PCR assay for genotyping of thiopurine intolerance-causing variant NUDT15 c.415C>T

2021 ◽  
pp. 153537022110265
Author(s):  
Xiaoyun Lian ◽  
Yanwei Li ◽  
Lan Li ◽  
Kaicheng U ◽  
Wenxia Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sanger Sequencing ◽  
Clinical Settings ◽  
Pcr Assay ◽  
Established Method ◽  
Single Tube ◽  
Taqman Probes ◽  
Autoimmune Conditions ◽  
Allele Specific

Thiopurines are commonly used in the treatment of acute lymphoblastic leukaemia and autoimmune conditions, can be limited by myelosuppression. The NUDT15 c.415C>T variant is strongly associated with thiopurine-induced myelosuppression, especially in Asians. The purpose of this study was to develop a fast and reliable genotyping method for NUDT15 c.415C>T and investigate the polymorphic distribution among different races in China. A single-tube multiplex real-time PCR assay for NUDT15 c.415C>T genotyping was established using allele-specific TaqMan probes. In 229 samples, the genotyping results obtained through the established method were completely concordant with those obtained by Sanger sequencing. The distributions of NUDT15 c.415C>T among 173 Han Chinese, 48 Miaos, 40 Kazakhs, and 40 Kirghiz were different, with allelic frequencies of 0.06, 0.02, 0.07, and 0, respectively. This method will provide a powerful tool for the implementation of the genotyping-based personalized prescription of thiopurines in clinical settings.

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