Standardisation and validation of an in-house quantitative real-time polymerase chain reaction (qPCR) assay for the diagnosis of Clostridioides difficile infection

2021 ◽  
pp. 106399
Author(s):  
Adriane C. Maestri ◽  
Sonia M. Raboni ◽  
Laura L. Cogo ◽  
Marisa V. Rossi ◽  
Keite S. Nogueira
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Qpcr Assay ◽  
Chain Reaction ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Polymerase Chain

scholarly journals Rapid Detection of the Vibrio cholerae ctx Gene in Food Enrichments Using Real-Time Polymerase Chain Reaction

2007 ◽  
Vol 90 (5) ◽  
pp. 1278-1283 ◽  
Author(s):  
Willis Fedio ◽  
George M Blackstone ◽  
Lynne Kikuta-Oshima ◽  
Chitra Wendakoon ◽  
Timothy H McGrath ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Vibrio Cholerae ◽  
Real Time ◽  
Bottled Water ◽  
Qpcr Assay ◽  
Second Phase ◽  
Chain Reaction ◽  
Isolation Frequency ◽  
Polymerase Chain ◽  
Static Conditions

Abstract A real-time polymerase chain reaction (qPCR) assay for the detection of the ctxA gene of toxigenic Vibrio cholerae (Vc) was validated against standard culture techniques. The first experimental phase determined optimal enrichment conditions for detection by culture and qPCR of Vc in shrimp, bottled water, milk, and potato salad. The conditions tested included temperature (35 and 42C), time (6 and 18 h), and effect of shaking (0 and 100 rpm). No definitive trends were found with enrichment temperature or shaking on Vc isolation frequency or detection by qPCR. Generally, Vc was detected by qPCR more frequently than Vc was isolated, but this difference was significant only in the 35C 6 h enrichment without shaking. In the second phase of experiments, shrimp, bottled water, milk, potato salad, and oysters were inoculated with each of 3 toxigenic Vc strains (Latin American O1 strain, an O139 strain, and an O1 strain from the U.S. Gulf Coast) and enriched under static conditions at 42C for 6 and 18 h. Overall, detection frequency of ctx by qPCR was 98 (88/90) and 100 (90/90) after 6 and 18 h enrichments, respectively, while Vc isolation frequency was 87 (78/90) and 83 (75/90) after 6 and 18 h, respectively. Toxigenic Vc can be detected by qPCR within an 8 h work day using the 6 h enrichment procedure, assuming an initial level of at least 12 colony-forming units/g; however, overnight enrichment may be necessary to detect lower levels. These data indicate that the qPCR assay for ctx is a more reliable, sensitive, and rapid alternative to standard Vc culture methods and is applicable to diverse food products.

scholarly journals Kinetics of polymerase chain reaction positivity in patients with Clostridioides difficile infection

2021 ◽  
Vol 14 ◽  
pp. 175628482110504
Author(s):  
Srishti Saha ◽  
Devvrat Yadav ◽  
Ryan Pardi ◽  
Robin Patel ◽  
Sahil Khanna ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Median Time ◽  
Median Duration ◽  
Treatment Initiation ◽  
Chain Reaction ◽  
Duration Of Treatment ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Polymerase Chain ◽  
Kinetics Of

Background: Polymerase chain reaction (PCR) is a sensitive test for diagnosing Clostridioides difficile infection (CDI) and could remain positive following resolution of CDI. The kinetics of PCR positivity following antibiotics for CDI is unknown. We studied this and whether it predicted CDI recurrence. Methods: Adults with CDI from October 2009 to May 2017 were included. Serial stool samples within 60 days of treatment were collected. Recurrent CDI was defined as diarrhea after interim symptom resolution with positive stool PCR within 56 or 90 days of treatment completion. Contingency table analysis was used to assess the risk of recurrence. Results: Fifty patients were included [median age: 51 (range = 20–86) years, 66% women]. Treatment given was metronidazole, 50% (25); vancomycin, 44% (22); both, 4% (2); and fidaxomicin, 2% (1). Median duration of treatment for all 50 patients was 14 (range = 8–60) days. The median duration of treatment in patients who got prolonged therapy (>14 days) ( n = 10) was 47 (range = 18–60) days. Median time to negative PCR was 9 (95% CI, 7–14) days from treatment initiation, which did not differ by antibiotics given ( p = 0.5). A positive PCR during or after treatment was associated with a higher risk of recurrence at 56 days ( p = 0.02) and at 90 days ( p = 0.009). Conclusion: The median time to negative PCR in CDI was 9 days from treatment initiation. The PCR positivity during or after treatment may be useful for recurrence prediction; larger studies are needed to validate these results.

scholarly journals Quantification of Xylella fastidiosa from Citrus Trees by Real-Time Polymerase Chain Reaction Assay

2002 ◽  
Vol 92 (10) ◽  
pp. 1048-1054 ◽  
Author(s):  
Antonio C. Oliveira ◽  
Marcelo A. Vallim ◽  
Camile P. Semighini ◽  
Welington L. Araújo ◽  
Gustavo H. Goldman ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Xylella Fastidiosa ◽  
Distribution Patterns ◽  
Cell Number ◽  
Qpcr Assay ◽  
Fluorescence Signal ◽  
Chain Reaction ◽  
Temporal And Spatial Distribution ◽  
Polymerase Chain

Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease of sweet orange cultivars in Brazil. Polymerase chain reaction (PCR)-based assays constitute the principal diagnostic method for detection of these bacteria. In this work, we established a real-time quantitative PCR (QPCR) assay to quantify X. fastidiosa in naturally and artificially infected citrus. The X. fastidiosa cell number detected in the leaves increased according to the age of the leaf, and bacteria were not detected in the upper midrib section in young leaves, indicating temporal and spatial distribution patterns of bacteria, respectively. In addition, the X. fastidiosa cell number quantified in leaves of ‘Pera’ orange and ‘Murcott’ tangor reflected the susceptible and resistant status of these citrus cultivars. None of the 12 endophytic citrus bacteria or the four strains of X. fastidiosa nonpathogenic to citrus that were tested showed an increase in the fluorescence signal during QPCR. In contrast, all 10 CVC-causing strains exhibited an increase in fluorescence signal, thus indicating the specificity of this QPCR assay. Our QPCR provides a powerful tool for studies of different aspects of the Xylella-citrus interactions, and can be incorporated into breeding programs in order to select CVC-resistant plants more quickly.

Impact of a Multiplexed Polymerase Chain Reaction Panel on Identifying Diarrheal Pathogens in Hematopoietic Cell Transplant Recipients

2019 ◽  
Vol 71 (7) ◽  
pp. 1693-1700 ◽  
Author(s):  
Wesley S Rogers ◽  
Lars F Westblade ◽  
Rosemary Soave ◽  
Stephen G Jenkins ◽  
Koen van Besien ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Hematopoietic Cell ◽  
Cell Transplant ◽  
Chain Reaction ◽  
Hematopoietic Cell Transplant ◽  
Clostridioides Difficile ◽  
Multiplexed Pcr ◽  
Clostridioides Difficile Infection ◽  
Polymerase Chain ◽  
The Impact

Abstract Background Diarrhea is common and associated with substantial morbidity among hematopoietic cell transplant (HCT) recipients, but the etiology is often not identified. Multiplexed polymerase chain reaction (PCR) assays increase the detection of diarrheal pathogens, but the impact of this technology in this population has not been evaluated. Methods Our center replaced stool cultures and other conventional microbiologic methods with the FilmArray Gastrointestinal Panel (GI PCR) in June 2016. We reviewed all adult patients who received an HCT from June 2014–May 2015 (pre–GI PCR, n = 163) and from June 2016–May 2017 (post–GI PCR, n = 182) and followed them for 1 year after transplantation. Clostridioides difficile infection was diagnosed by an independent PCR test in both cohorts. Results The proportion of patients with ≥1 identified infectious diarrheal pathogen increased from 25% to 37% after implementation of GI PCR (P = .01). Eight patients (5%) in the pre–GI PCR cohort tested positive for a pathogen other than C. difficile versus 49 patients (27%) in the post–GI PCR cohort (P < .001). The most common non–C. difficile diarrheal pathogens in the post–GI PCR cohort were enteropathogenic Escherichia coli (n = 14, 8%), norovirus (n = 14, 8%), and Yersinia enterocolitica (n = 7, 4%). The percentage of diarrheal episodes with an identified infectious etiology increased from 14% to 23% (P = .001). Median total costs of stool testing per patient did not increase (pre: $473; post: $425; P = .25). Conclusions Infectious etiologies of diarrhea were identified in a higher proportion of HCT recipients after replacing conventional stool testing with a multiplexed PCR assay, without an increase in testing costs.

scholarly journals Peptide Nucleic Acid (PNA)-Enhanced Specificity of a Dual-Target Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Assay for the Detection and Differentiation of SARS-CoV-2 from Related Viruses

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 775
Author(s):  
Won-Suk Choi ◽  
Ju Hwan Jeong ◽  
Halcyon Dawn G. Nicolas ◽  
Sol Oh ◽  
Khristine Joy C. Antigua ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Real Time ◽  
Peptide Nucleic Acid ◽  
Detection Method ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Dual Target

The threat posed by coronaviruses to human health has necessitated the development of a highly specific and sensitive viral detection method that could differentiate between the currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related coronaviruses (SARSr-CoVs). In this study, we developed a peptide nucleic acid (PNA)-based real-time quantitative polymerase chain reaction (RT-qPCR) assay targeting the N gene to efficiently discriminate SARS-CoV-2 from other SARSr-CoVs in human clinical samples. Without compromising the sensitivity, this method significantly enhanced the specificity of SARS-CoV-2 detection by 100-fold as compared to conventional RT-qPCR. In addition, we designed an RT-qPCR method for the sensitive and universal detection of ORF3ab-E genes of SARSr-CoV with a limit of detection (LOD) of 3.3 RNA copies per microliter. Thus, the developed assay serves as a confirmative dual-target detection method. Our PNA-mediated dual-target RT-qPCR assay can detect clinical SARS-CoV-2 samples in the range of 18.10–35.19 Ct values with an 82.6–100% detection rate. Furthermore, our assay showed no cross-reactions with other coronaviruses such as human coronaviruses (229E, NL63, and OC43) and Middle East respiratory syndrome coronavirus, influenza viruses (Type B, H1N1, H3N2, HPAI H5Nx, and H7N9), and other respiratory disease-causing viruses (MPV, RSV A, RSV B, PIV, AdV, and HRV). We, thus, developed a PNA-based RT-qPCR assay that differentiates emerging pathogens such as SARS-CoV-2 from closely related viruses such as SARSr-CoV and allows diagnosis of infections related to already identified or new coronavirus strains.

scholarly journals Changes in the Association Between Diagnostic Testing Method, Polymerase Chain Reaction Ribotype, and Clinical Outcomes From Clostridioides difficile Infection: One Institution’s Experience

2020 ◽  
Author(s):  
Anitha Menon ◽  
D Alex Perry ◽  
Jonathan Motyka ◽  
Shayna Weiner ◽  
Alexandra Standke ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Testing ◽  
Adverse Outcomes ◽  
Clinical Severity ◽  
Diagnostic Strategy ◽  
Chain Reaction ◽  
Ribotype 027 ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Polymerase Chain

Abstract Background In Clostridioides difficile infection (CDI), the relationship between clinical, microbial, and temporal/epidemiological trends, disease severity and adverse outcomes is incompletely understood. In a follow-up to our study from 2010–2013, we evaluate stool toxin levels and C. difficile polymerase chain reaction (PCR) ribotypes. We hypothesized that elevated stool toxins and infection with ribotype 027 associate with adverse outcomes. Methods In 565 subjects at the University of Michigan with CDI diagnosed by positive testing for toxins A/B by enzyme immunoassay (EIA) or PCR for the tcdB gene, we quantified stool toxin levels via a modified cell cytotoxicity assay (CCA), isolated C. difficile by anaerobic culture, and performed PCR ribotyping. Severe CDI was defined by Infectious Diseases Society of America (IDSA) criteria, and primary outcomes were all-cause 30-day mortality and a composite of colectomy, intensive care unit admission, and/or death attributable to CDI within 30 days. Analyses included bivariable tests and logistic regression. Results 199 samples were diagnosed by EIA; 447 were diagnosed by PCR. Toxin positivity associated with IDSA severity but not primary outcomes. In 2016, compared with 2010–2013, ribotype 106 newly emerged, accounting for 10.6% of strains, ribotype 027 fell from 16.5% to 9.3%, and ribotype 014–027 remained stable at 18.9%. Ribotype 014–020 associated with IDSA severity and 30-day mortality (P = .001). Conclusions Toxin positivity by EIA and CCA associated with IDSA severity but not with subsequent adverse outcomes. The molecular epidemiology of C. difficile has shifted, which may have implications for the optimal diagnostic strategy for and clinical severity of CDI.

Detection of Nucleophosmin 1 Mutations by Quantitative Real-Time Polymerase Chain Reaction Versus Capillary Electrophoresis: A Comparative Study

2011 ◽  
Vol 135 (8) ◽  
pp. 994-1000 ◽  
Author(s):  
Fareed H Barakat ◽  
Rajyalakshmi Luthra ◽  
C. Cameron Yin ◽  
Bedia A Barkoh ◽  
Seema Hai ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Capillary Electrophoresis ◽  
Real Time ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Qpcr Assay ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Minimal Residual

Context.—Nucleophosmin 1 (NPM1) is the most commonly mutated gene in acute myeloid leukemia. Detection of NPM1 mutations is useful for stratifying patients for therapy, predicting prognosis, and assessing for minimal residual disease. Several methods have been developed to rapidly detect NPM1 mutations in genomic DNA and/or messenger RNA specimens. Objective.—To directly compare a quantitative real-time polymerase chain reaction (qPCR) assay with a widely used capillary electrophoresis assay for detecting NPM1 mutations. Design.—We adopted and modified a qPCR assay designed to detect the 6 most common NPM1 mutations and performed the assay in parallel with capillary electrophoresis assay in 207 bone marrow aspirate or peripheral blood samples from patients with a range of hematolymphoid neoplasms. Results.—The qPCR assay demonstrated a higher analytical sensitivity than the capillary electrophoresis 1/1000 versus 1/40, respectively. The capillary electrophoresis assay generated 10 equivocal results that needed to be repeated, whereas the qPCR assay generated only 1 equivocal result. After test conditions were optimized, the qPCR and capillary electrophoresis methods produced 100% concordant results, 85 positive and 122 negative. Conclusions.—Given the higher analytical sensitivity and specificity of the qPCR assay, that assay is less likely to generate equivocal results than the capillary electrophoresis assay. Moreover, the qPCR assay is quantitative, faster, cheaper, less prone to contamination, and well suited for monitoring minimal residual disease.

Sign in / Sign up

Export Citation Format

Share Document