A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything

Králík, Petr; Ricchi, M.

doi:10.3389/fmicb.2017.00108

Cited by 714 publications

(605 citation statements)

References 35 publications

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“…There are several PCR modifications that can help to increase the sensitivity and specificity of detection, including real‐time PCR that provides fast detection and quantitation of target DNA . However, this procedure has several disadvantages: high equipment cost, high technical skill, increased risk of false‐negative results due to the human error as a result of improper assay development, and incorrect data analysis . The simpler alternative is nested PCR that involves two rounds of PCR.…”

Section: Introductionmentioning

confidence: 99%

Diagnostic reliability of nested PCR depends on the primer design and threshold abundance ofHelicobacter pyloriin biopsy, stool, and saliva samples

et al. 2020

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Background The aim of this work was to find a reliable nested PCR for the detection of Helicobacter pylori in biopsy, stool, and saliva specimens. Materials and Methods Novel nested PCR was elaborated and validated on 81 clinical biopsy, stool, and saliva samples from the same individual and compared to available H pylori assays: histology, rapid urease test (RUT), stool antigen test (SAT), 13C‐urea breath test (UBT). Results The efficiency and selectivity of 17 published nested polymerase chain reactions (PCR) available for Helicobacter pylori detection were re‐evaluated. Most of them had serious limitations and mistakes in primer design. Hence, we elaborated a nested PCR for the unambiguous identification of H pylori in biopsy, stool, and saliva, using primers targeted to variable regions of the 16S ribosomal RNA (rRNA) gene. Moreover, we determined the detection limit by adding a known number of cells. This number was as low as 0.5 cells in a PCR vial, but due to the DNA isolation procedures, it required 1‐5 × 103 cells/g or ml of specimen. The sensitivity for nested PCR from stomach biopsies was on the same scale as 13C‐UBT (93.8%), but it was much lower in amplifications from stool (31.3%). Sequencing of all obtained PCR products exclusively confirmed H pylori‐specific DNA sequences. Conclusions Elaborated nested PCR assay can serve as an auxiliary method for controversial samples (patients with bleeding or taking proton‐pump inhibitor) in laboratories with basic equipment. The sensitivity and specificity for the amplification from gastric biopsies was almost like 13C‐UBT. Despite the good sensitivity, the threshold occurrence and the ability to survive in the oral cavity aside from and independent of the stomach is the reason why H pylori DNA cannot be reliably detected in saliva, stool, and some biopsy samples.

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Section: Introductionmentioning

confidence: 99%

Diagnostic reliability of nested PCR depends on the primer design and threshold abundance ofHelicobacter pyloriin biopsy, stool, and saliva samples

et al. 2020

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“…Non-standard real-time PCR analyses can produce false-positive and falsenegative results based on the height of the threshold and the slope of the amplification curve. Thus, standard documentation and procedures are needed to determine the basic functional parameters of real-time PCR in the field of routine microbiological diagnosis (60).…”

Section: Resultsmentioning

confidence: 99%

“…More generally, the amplification reaction where Nn is the number of PCR amplifications after n cycles, N0 is the initial number of template replicates in the sample, E is the PCR efficiency, and n is the number of cycles (60). This equation is based on the assumption that there is initially one template in the reaction and the PCR efficiency is 100%.…”

Section: All Detection Methods Rely On Fret (55) Among These Detectimentioning

confidence: 99%

“…This equation is based on the assumption that there is initially one template in the reaction and the PCR efficiency is 100%. In other words, as the PCR efficiency is 100%, accurate data can be obtained (60). To do so, the standard control used to create the standard curve in real-time PCR must be accurate.…”

Section: All Detection Methods Rely On Fret (55) Among These Detectimentioning

confidence: 99%

“…According to the MIQE guidelines and various performance evaluation guidelines (60,80), the developed real-time PCR method should be validated based on analysis performance and clinical trial evaluation.…”

Section: Pcr-based Techniquesmentioning

confidence: 99%

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Diagnosis of Viral Infection Using Real-time Polymerase Chain Reaction

Hwang

Ahn²,

Nam

2018

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The laboratory-based diagnosis of viral infection has been evolving over the years, to increase objectivity, accuracy, and sensitivity via the continuous development of various technologies. Cell culture, which is one of the methods used for the diagnosis of viral infection, is a "gold-standard" approach; however, it is time consuming and is associated with a high risk of contamination. To overcome these shortcomings, molecular biology methods, such as conventional polymerase chain reaction (cPCR), real-time PCR, and sequencing, have been used recently for virus diagnosis. Realtime PCR has higher accuracy and sensitivity compared with cPCR. Moreover, realtime PCR can quantify viral nucleic acids by confirming the amplification using the threshold cycle, which is the initial amplification point. Real-time PCR applications for the detection of various types of viruses in clinical settings should be based on the use of appropriate samples, nucleic acid extraction according to virus characteristics, and selection of diagnostic methods using sensitivity and specificity targets. In addition, the implementation of real-time PCR requires to evaluate the performance of the test protocol by measuring sensitivity, specificity, accuracy, and reproducibility. The verified real-time PCR method is an easy, fast, and accurate method for monitoring the diagnosis and treatment outcomes in a clinical setting. In this review, we summarize the characteristics of the typical diagnostic methods for viral infection, especially of the advanced real-time PCR method, to detect human pathogenic viruses.

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Serum and urine nucleic acid screening tests for BK polyomavirus-associated nephropathy in kidney and kidney-pancreas transplant recipients

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et al. 2024

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A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything

Cited by 714 publications

References 35 publications

Diagnostic reliability of nested PCR depends on the primer design and threshold abundance ofHelicobacter pyloriin biopsy, stool, and saliva samples

Diagnostic reliability of nested PCR depends on the primer design and threshold abundance ofHelicobacter pyloriin biopsy, stool, and saliva samples

Diagnosis of Viral Infection Using Real-time Polymerase Chain Reaction

Serum and urine nucleic acid screening tests for BK polyomavirus-associated nephropathy in kidney and kidney-pancreas transplant recipients

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