Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

Nagy, Alexander; Vitásková, Eliška; Černíková, Lenka; Křivda, Vlastimil; Jiřincová, Helena; Sedlák, Kamil; Horníčková, Jitka; Havlíčková, Martina

doi:10.1038/srep41392

Cited by 36 publications

(32 citation statements)

References 29 publications

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“…TaqMan qPCR is a powerful tool used in the microbial diagnosis of samples from different sources [19]. Hence, in this study, we developed a multiplex TaqMan qPCR (MTq-PCR) with three novel phylum-specific TaqMan probes to determine the proportions of the three dominant phyla in the gut microbiota of UC patients (n = 6) and healthy subjects (n = 6), namely Bacteroidetes, Firmicutes, and Proteobacteria.…”

Section: Discussionmentioning

confidence: 99%

“…However, these methods are not able to simultaneously quantify different phyla in a single tube. TaqMan PCR is a powerful tool for the simultaneous detection and quantification of microbes in samples from different sources [18][19][20]. The relative composition of the three major phyla in the human gut, Bacteroidetes, Firmicutes, and Proteobacteria, has been proposed as a potential diagnostic tool for UC [9,21].…”

Section: Introductionmentioning

confidence: 99%

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In Situ Profiling of the Three Dominant Phyla Within the Human Gut Using TaqMan PCR for Pre-Hospital Diagnosis of Gut Dysbiosis

Tagele

Pham

et al. 2020

IJMS

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A microbial imbalance called dysbiosis leads to inflammatory bowel disease (IBD), which can include ulcerative colitis (UC). Fecal microbiota transplantation (FMT), a novel therapy, has recently been successful in treating gut dysbiosis in UC patients. For the FMT technique to be successful, the gut microbiota of both the healthy donors and UC patients must be characterized. For decades, next-generation sequencing (NGS) has been used to analyze gut microbiota. Despite the popularity of NGS, the cost and time constraints make it difficult to use in emergency services and activities related to the periodic monitoring of microbiota profile alterations. Hence, in this study, we developed a multiplex TaqMan qPCR assay (MTq-PCR) with novel probes to simultaneously determine the relative proportions of the three dominant microbial phyla in the human gut: Bacteroidetes, Firmicutes, and Proteobacteria. The relative proportions of the three phyla in fecal samples of either healthy volunteers or UC patients were similar when assessed NGS and the MTq-PCR. Thus, our MTq-PCR assay could be a practical microbiota profiling alternative for diagnosing and monitoring gut dysbiosis in UC patients during emergency situations, and it could have a role in screening stool from potential FMT donors.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

In Situ Profiling of the Three Dominant Phyla Within the Human Gut Using TaqMan PCR for Pre-Hospital Diagnosis of Gut Dysbiosis

Tagele

Pham

et al. 2020

IJMS

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“…Therefore, optimization of primers and reaction kinetics are needed, including DNA gel analysis, during validation of these assays. The latter issue can be to some extent minimized or eliminated through the use of another commonly used DNA detection system, the TaqMan probe chemistry 15 . In this case, another layer of specificity is added whereby after DNA amplification the TaqMan probe binds to the PCR product of interest (avoiding other non‐specific amplicons).…”

Section: Methods In Molecular Diagnosticsmentioning

confidence: 99%

“…In this case, another layer of specificity is added whereby after DNA amplification the TaqMan probe binds to the PCR product of interest (avoiding other non‐specific amplicons). Once the probe binds, the DNA polymerase releases a fluorescent dye (attached to the probe) utilizing its intrinsic 5ʹ→3ʹ exonuclease activity 15 . Overall, there are advantages and disadvantages to consider when choosing RT‐PCR dyes—for an in‐depth discussion in this topic, we refer the reader to other reviews 3…”

Section: Methods In Molecular Diagnosticsmentioning

confidence: 99%

Clinical molecular genetics evaluation in women with reproductive failures

Bilal

Katara

Dambaeva

et al. 2020

American J Rep Immunol

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Molecular diagnostics is a rapidly growing branch of the clinical laboratory and has accelerated the advance of personalized medicine in the fields of pharmacogenomics, pharmacogenetics, and nutrigenomics. The versatility of molecular biology allows it to be effective in several medical fields that include reproduction, immunogenetics, and virology. Implementation of molecular and sequencing technology in reproductive medicine can add another layer of understanding to better define the causes behind infertility and recurrent reproductive loss. In the following, we examine current molecular methods for probing factors behind reproductive pregnancy loss including reverse transcription polymerase chain reaction and next generation sequencing (NGS). We review several current and potential genetic (DNA) and transcriptional (RNA)‐based parameters in women with infertility that can be significant in diagnosis and treatment. These molecular factors can be inferred either from genomic DNA or RNA locally within the endometrium. Furthermore, we consider infection‐based abnormalities such as human herpesvirus‐6 and severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). Finally, we present future directions as well as data demonstrating the potential role of human endogenous retroviruses in pregnancy loss. We hope these discussions will assist the clinician in delineating some of the intricate molecular factors that can contribute to infertility and recurrent reproductive failures.

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“…Real-time PCR assay has minimized these inconveniences; it does not require additional confirmatory steps and can report more accurate result in relatively shorter time than other conventional NAATs. More recently, TaqMan probe method (Applied Biosystems, Foster City, CA, USA) has been developed that enables multiple target detections retaining the accuracy and rapid performance of real time PCR assay [ 24 25 ]. Therefore, it has been frequently adopted for TB diagnosis along with many other related assays commercially available [ 26 ].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of EZplex MTBC/NTM Real-Time PCR kit: diagnostic accuracy and efficacy in vaccination

Lee

Hwang²,

Ahn³

et al. 2018

Clin Exp Vaccine Res

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PurposeTuberculosis (TB) is mainly caused by Mycobacterium tuberculosis, which is a pathogenic mycobacterial species grouped under Mycobacterium tuberculosis complex (MTBC) with four other pathogenic mycobacterial species. The mycobacteria not included in MTBC are known as nontuberculous mycobacteria (NTM), and cause several pulmonary diseases including pneumonia. Currently, NTM occurrences in TB-suspected respiratory specimens have increased, due to which, precise detection of MTBC and NTM is considered critical for the diagnosis and vaccination of TB. Among the various methods available, real-time PCR is frequently adopted for MTBC/NTM detection due to its rapidness, accuracy, and ease of handling. In this study, we evaluated a new real-time PCR kit for analytical and clinical performance on sputum, bronchial washing, and culture specimens.Materials and MethodsFor assessing its analytical performance, limit of detection (LOD), reactivity, and repeatability test were performed using DNA samples. To evaluate clinical performance, 612 samples were collected and clinically tested at a tertiary hospital.ResultsLOD was confirmed as 0.584 copies/µL for MTBC and 47.836 copies/µL for NTM by probit analysis (95% positive). For the reactivity test, all intended strains were detected and, in the repeatability test, stable and steady results were confirmed with coefficient of variation ranging from 0.36 to 1.59. For the clinical test, sensitivity and specificity were 98.6%–100% and 98.8%–100% for MTBC and NTM, respectively.ConclusionThe results proved the usefulness of the kit in TB diagnosis. Furthermore, it could be adopted for the assessment of vaccine efficacy.

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Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

Cited by 36 publications

References 29 publications

In Situ Profiling of the Three Dominant Phyla Within the Human Gut Using TaqMan PCR for Pre-Hospital Diagnosis of Gut Dysbiosis

In Situ Profiling of the Three Dominant Phyla Within the Human Gut Using TaqMan PCR for Pre-Hospital Diagnosis of Gut Dysbiosis

Clinical molecular genetics evaluation in women with reproductive failures

Evaluation of EZplex MTBC/NTM Real-Time PCR kit: diagnostic accuracy and efficacy in vaccination

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