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.

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.

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.

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

scholarly journals Clinical Validation of Multiplex Real-Time PCR Assays for Detection of Bacterial Meningitis Pathogens

2011 ◽  
Vol 50 (3) ◽  
pp. 702-708 ◽  
Author(s):  
X. Wang ◽  
M. J. Theodore ◽  
R. Mair ◽  
E. Trujillo-Lopez ◽  
M. du Plessis ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Validation ◽  
Pcr Assays

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 656
Author(s):  
Konstantin Tanida ◽  
Andreas Hahn ◽  
Kirsten Alexandra Eberhardt ◽  
Egbert Tannich ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Real Time ◽  
Indigenous People ◽  
Real Time Pcr ◽  
Latent Class ◽  
Enterocytozoon Bieneusi ◽  
Enteric Disease ◽  
Immunosuppressed Patients ◽  
Study Population ◽  
Stool Samples ◽  
Pcr Assays

Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen’s kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied.

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