Implementation of a process internal control to monitor the performance of real-time PCR assays for the detection of Vibrio cholerae in water

2013 ◽  
Vol 13 (3) ◽  
pp. 761-768
Author(s):  
V. M. Ntema ◽  
T. G. Barnard
Keyword(s):  
Process Control ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Tap Water ◽  
E Coli ◽  
Negative Results ◽  
Alkaline Peptone Water ◽  
Pcr Assays

The process control utilised in this study was an Escherichia coli (E. coli-GFP) strain carrying a chromosomally integrated copy of the green fluorescent protein (gfp) gene of the jelly fish Aequorea victoria. Application of the process control involved spiking samples with the E. coli-GFP containing cells and detecting gfp using real-time polymerase chain reaction (PCR). The process control was implemented in the context of two multiplex real-time PCR assays. The first multiplex targeted the ctxA, hlyA and gfp genes, while the second targeted the gfp, O1-rfb, and O139-rfb loci. The detection limits for both the multiplex real-time PCR assays were 20 CFU (colony forming units) per reaction. This method was evaluated using spiked and unspiked Alkaline Peptone Water (APW) enrichments of sewage, dam, catchment and tap water. The internal process control showed no substantial inhibition in the APW enriched water samples, assuring the integrity of negative results. The results from this study showed that the use of the E. coli-GFP strain as a process internal control could provide quality assurance and increase the reliable interpretation of real-time PCR results.

DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

2019 ◽  
pp. 60-66
Author(s):  
Viet Quynh Tram Ngo ◽  
Thi Ti Na Nguyen ◽  
Hoang Bach Nguyen ◽  
Thi Tuyet Ngoc Tran ◽  
Thi Nam Lien Nguyen ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Type B ◽  
E Coli ◽  
Pcr Assays ◽  
Set Up ◽  
Etiological Agents ◽  
Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

Potential pitfalls in the quantitative molecular detection ofEscherichia coliO157:H7 in environmental matrices

2006 ◽  
Vol 52 (5) ◽  
pp. 482-488 ◽  
Author(s):  
Rebekka R.E Artz ◽  
Lisa M Avery ◽  
Davey L Jones ◽  
Ken Killham
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Soil System ◽  
E Coli ◽  
Animal Wastes ◽  
Pcr Assays

The detection sensitivity and potential interference factors of a commonly used assay based on real-time polymerase chain reaction (PCR) for Escherichia coli O157:H7 using eae gene-specific primers were assessed. Animal wastes and soil samples were spiked with known replicate quantities of a nontoxigenic strain of E. coli O157:H7 in a viable or dead state and as unprotected DNA. The detection sensitivity and accuracy of real-time PCR for E. coli O157:H7 in animal wastes and soil is low compared to enrichment culturing. Nonviable cells and unprotected DNA were shown to produce positive results in several of the environmental samples tested, leading to potential overestimates of cell numbers due to prolonged detection of nonviable cells. This demonstrates the necessity for the specific calibration of real-time PCR assays in environmental samples. The accuracy of the eae gene–based detection method was further evaluated over time in a soil system against an activity measurement, using the bioluminescent properties of an E. coli O157:H7 Tn5luxCDABE construct. The detection of significant numbers of viable but nonculturable (VBNC) as well as nonviable and possibly physically protected cells as shown over a period of 90 days further complicates the use of real-time PCR assays for quick diagnostics in environmental samples and infers that enrichment culturing is still required for the final verification of samples found positive by real-time PCR methods.Key words: Escherichia coli O157:H7, real-time PCR, animal waste, soil, VBNC.

Routine use of duplex real-time PCR assays including a commercial internal control for molecular diagnosis of opportunistic DNA virus infections

2012 ◽  
Vol 185 (1) ◽  
pp. 136-141 ◽  
Author(s):  
Sonia Burrel ◽  
Christelle Fovet ◽  
Christel Brunet ◽  
Lydia Ovaguimian ◽  
Nathalie Hamm ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Diagnosis ◽  
Internal Control ◽  
Real Time Pcr ◽  
Virus Infections ◽  
Dna Virus ◽  
Pcr Assays

scholarly journals Simple Technique for Internal Control of Real-Time Amplification Assays

2004 ◽  
Vol 50 (5) ◽  
pp. 819-825 ◽  
Author(s):  
Siegfried Burggraf ◽  
Bernhard Olgemöller
Keyword(s):  
Melting Point ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
False Negative ◽  
Hybridization Probe ◽  
Melting Point Analysis ◽  
Point Analysis ◽  
Pcr Assays ◽  
Time Amplification

Abstract Background: In real-time PCR assays, the most accurate way to identify false-negative results, e.g., those caused by PCR inhibitors, is to add to samples an internal control that will be coamplified with the target (e.g., pathogen) DNA. Current internal control procedures, however, which usually involve the introduction of a DNA fragment, are complex, time-consuming, and expensive. Methods: Single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time PCR assays. Mismatches were included in the probe-binding region of the internal control oligonucleotide (ICO) to prevent probe–control hybridization during the fluorescence acquisition step of the PCR. Amplified ICOs were detected by melting point analysis. ICOs could be added directly to the sample material before DNA extraction. Results: To demonstrate the feasibility of the new approach, we designed ICOs for the LightCycler hybridization probe assays for Mycobacterium tuberculosis complex, hepatitis B virus, herpes simplex virus, and varicella zoster virus. In each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis. Conclusions: A single-stranded oligonucleotide that mimics the target region of the pathogen but is clearly distinguishable from the target during melting point analysis can serve as a simple, cost-effective internal control for real-time amplification assays. Such control oligonucleotides are easy to design and inexpensive. A costly second probe system is not necessary. Moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications.

scholarly journals Intimin Gene (eae) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces

2013 ◽  
Vol 80 (3) ◽  
pp. 1177-1184 ◽  
Author(s):  
Delphine Bibbal ◽  
Estelle Loukiadis ◽  
Monique Kérourédan ◽  
Carine Peytavin de Garam ◽  
Franck Ferré ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Specific Detection ◽  
Content Type ◽  
E Coli ◽  
Cattle Feces ◽  
Pcr Assays

ABSTRACTShiga toxin-producingEscherichia coli(STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targetedeaesubtypes. The simultaneous presence ofstx,eae, and one of the five O group markers was found in 58.0% of the samples, and the five targetedstxpluseaeplus O genetic combinations were detected 143 times. However, taking into consideration the association betweeneaesubtypes and O group markers, the resultingstxpluseaesubtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22E. colistrains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive forstx,eaeand an O group marker, but that were negative for the correspondingeaesubtype, were successful. Characterization of the 24E. coliisolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenicE. coli(aEPEC). Finally, the more discriminatingeaesubtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

scholarly journals Development and application of a canine endogenous internal positive control for use in real-time PCR assays

2018 ◽  
Vol 30 (5) ◽  
pp. 789-792 ◽  
Author(s):  
Joseph J. Modarelli ◽  
Pamela J. Ferro ◽  
Maria D. Esteve-Gasent
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Positive Control ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Negative Results ◽  
False Negative Results ◽  
Internal Positive Control ◽  
Pcr Assays

Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats.

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.

Application of real-time PCR and RT-PCR assays for the detection and quantitation of VT 1 and VT 2 toxin genes in E. coli O157:H7

2004 ◽  
Vol 18 (2) ◽  
pp. 123-132 ◽  
Author(s):  
J Fitzmaurice ◽  
M Glennon ◽  
G Duffy ◽  
J.J Sheridan ◽  
C Carroll ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rt Pcr ◽  
E Coli ◽  
Toxin Genes ◽  
Pcr Assays
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