scholarly journals Rapid Real-Time Polymerase Chain Reaction for Salmonella Serotyping Based on Novel Unique Gene Markers by Pangenome Analysis

2021 ◽  
Vol 12 ◽  
Author(s):  
Seung-Min Yang ◽  
Eiseul Kim ◽  
Dayoung Kim ◽  
Hyeon-Be Kim ◽  
Jiwon Baek ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Specific Gene ◽  
Chain Reaction ◽  
Gene Markers ◽  
Unique Gene ◽  
High Prevalence ◽  
Polymerase Chain

An accurate diagnostic method for Salmonella serovars is fundamental to preventing the spread of associated diseases. A diagnostic polymerase chain reaction (PCR)-based method has proven to be an effective tool for detecting pathogenic bacteria. However, the gene markers currently used in real-time PCR to detect Salmonella serovars have low specificity and are developed for only a few serovars. Therefore, in this study, we explored the novel unique gene markers for 60 serovars that share similar antigenic formulas and show high prevalence using pangenome analysis and developed a real-time PCR to detect them. Before exploring gene markers, the 535 Salmonella genomes were evaluated, and some genomes had serovars different from the designated serovar information. Based on these analyses, serovar-specific gene markers were explored. These markers were identified as genes present in all strains of target serovar genomes but absent in strains of other serovar genomes. Serovar-specific primer pairs were designed from the gene markers, and a real-time PCR method that can distinguish between 60 of the most common Salmonella serovars in a single 96-well plate assay was developed. As a result, real-time PCR showed 100% specificity for 199 Salmonella and 29 non-Salmonella strains. Subsequently, the method developed was applied successfully to both strains with identified serovars and an unknown strain, demonstrating that real-time PCR can accurately detect serovars of strains compared with traditional serotyping methods, such as antisera agglutination. Therefore, our method enables rapid and economical Salmonella serotyping compared with the traditional serotyping method.

scholarly journals DETEKSI CLOSTRIDIUM DIFFICILE TOKSIGENIK MENGGUNAKAN UJI CEPAT TOKSIN DAN REAL TIME POLYMERASE CHAIN REACTION (Toxigenic Clostridium Difficile Detection Using Toxin Rapid Test and Real Time Polymerase Chain Reaction)

2018 ◽  
Vol 22 (1) ◽  
pp. 22
Author(s):  
Ika Yasma Yanti ◽  
Dalima Ari Wahono Astrawinata
Keyword(s):  
Polymerase Chain Reaction ◽  
Clostridium Difficile ◽  
Antibiotic Therapy ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Test ◽  
Chain Reaction ◽  
Chi Square ◽  
Polymerase Chain ◽  
Clostridium Difficile Associated Diarrhea

Toxigenic Clostridium difficile infection, causing a Pseudo Membrane Colitis (PMC) and Clostridium Difficile Associated Diarrhea(CDAD) has increased sharply. The largest risk factor is the use of antibiotics. The purpose of this study was to know how to determinethe prevalence and characteristics of subjects with Toxigenic Clostridium difficile and to assess the ability of the toxin rapid test comparedto real-time PCR. Ninety adult subjects with antibiotic therapy more than two (2) weeks were enrolled in this study. The results of toxinrapid test and real-time PCR were presented in a 2x2 table, statistical test used was Chi square. The prevalence of Toxigenic Clostridiumdifficile based on the toxin rapid test and by real-time PCR was 27.3% and 37.5%, respectively. There were significant differences betweenstool consistency and number of antibiotics used with the detection of Toxigenic Clostridium difficile. There was a relationship betweenthe duration of antibiotic therapy with the detection of Toxigenic Clostridium difficile using real-time PCR (p=0.010, RR=2.116). Thesensitivity, specificity, PPV, NPV, PLR and NLR rapid test against real-time PCR were 69.7%; 98.2%; 95.8%; 84.4%; 39.2 and 0.31,respectively. This study concluded that the prevalence of Clostridium difficile in RSCM was higher compared to that in Malaysia, Thailandand India; the subjects with antibiotic therapy for more than four (4) weeks had a double risk to have Toxigenic Clostridium difficilethan subjects with antibiotic therapy for less than that time (4 weeks). Thus, in this study, toxin rapid test could be used as a tool todetect Toxigenic Clostridium difficile.

scholarly journals Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

2021 ◽  
Author(s):  
Rajeev Kumar Jain ◽  
Nagaraj Perumal ◽  
Rakesh Shrivastava ◽  
Kamlesh Kumar Ahirwar ◽  
Jaya Lalwani ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Specific Gene ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Gene Target ◽  
Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

THE DETECTION OF THE GENUS LISTERIA PATHOGENIC BACTERIA BY REAL-TIME POLYMERASE CHAIN REACTION

2020 ◽  
pp. 117-125
Author(s):  
Elena V. Sokolova ◽  
Ekaterina K. Psareva ◽  
Irina Yu. Egorova ◽  
Pavel A. Zhurilov ◽  
Evgeny A. Potemkin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathogenic Bacteria ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals In Planta Distribution of ‘Candidatus Liberibacter asiaticus’ as Revealed by Polymerase Chain Reaction (PCR) and Real-Time PCR

2008 ◽  
Vol 98 (5) ◽  
pp. 592-599 ◽  
Author(s):  
Satyanarayana Tatineni ◽  
Uma Shankar Sagaram ◽  
Siddarame Gowda ◽  
Cecile J. Robertson ◽  
William O. Dawson ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Citrus Tree ◽  
Candidatus Liberibacter Asiaticus ◽  
In Planta ◽  
Chain Reaction ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic α-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of ‘Candidatus Liberibacter asiaticus,’ respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that ‘Ca. Liberibacter asiaticus’ was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/μg of total DNA in different tissues. A relatively high concentration of ‘Ca. Liberibacter asiaticus’ was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that ‘Ca. Liberibacter asiaticus’ was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

scholarly journals Real-Time Polymerase Chain Reaction for One-Hour On-Site Diagnosis of Pierce's Disease of Grape in Early Season Asymptomatic Vines

2002 ◽  
Vol 92 (7) ◽  
pp. 721-728 ◽  
Author(s):  
N. W. Schaad ◽  
D. Opgenorth ◽  
P. Gaush
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Xylella Fastidiosa ◽  
Molecular Techniques ◽  
Plant Diseases ◽  
Pierce's Disease ◽  
Chain Reaction ◽  
Pierce’S Disease ◽  
Polymerase Chain

Molecular-based techniques, such as polymerase chain reaction (PCR), can reduce the time needed for diagnosis of plant diseases when compared with classical isolation and pathogenicity tests. However, molecular techniques still require 2 to 3 days to complete. To the best of our knowledge, we describe for the first time a real-time PCR technique using a portable Smart Cycler for one-hour on-site diagnosis of an asymptomatic plant disease. Pierce's disease (PD) of grape, caused by the fastidious bacterium Xylella fastidiosa, causes serious losses in grapes in California and the southeastern United States. The disease has been difficult to diagnose because typical leaf scorching symptoms do not appear until late (June and after) in the season and the organism is very difficult to isolate early in the season. Sap and samples of macerated chips of secondary xylem from trunks of vines were used in a direct real-time PCR without extraction of DNA. Using two different sets of primers and probe, we diagnosed PD in 7 of 27 vines (26%) from four of six vineyards sampled 10 to 12 days after bud break in Kern, Tulare, and Napa counties of California. The diagnosis was confirmed by isolation of Xylella fastidiosa from two of the original PCR positive samples and later from symptomatic leaf petioles of four out of four vines from one vineyard that were originally PCR positive.

Real-Time Polymerase Chain Reaction Detection of Asymptomatic Clostridium difficile Colonization and Rising C. difficile–Associated Disease Rates

2014 ◽  
Vol 35 (6) ◽  
pp. 667-673 ◽  
Author(s):  
Hoonmo L. Koo ◽  
John N. Van ◽  
Meina Zhao ◽  
Xunyan Ye ◽  
Paula A. Revell ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clostridium Difficile ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Detection ◽  
Adult Patients ◽  
Incidence Density ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Polymerase Chain

Objective.To evaluate the accuracy of real-time polymerase chain reaction (PCR) for Clostridium difficile–associated disease (CDAD) detection, after hospital CDAD rates significantly increased following real-time PCR initiation for CDAD diagnosis.Design.Hospital-wide surveillance study following examination of CDAD incidence density rates by interrupted time series design.Setting.Large university-based hospital.Participants.Hospitalized adult patients.Methods.CDAD rates were compared before and after real-time PCR implementation in a university hospital and in the absence of physician and infection control practice changes. After real-time PCR introduction, all hospitalized adult patients were screened for C. difficile by testing a fecal specimen by real-time PCR, toxin enzyme-linked immunosorbent assay, and toxigenic culture.Results.CDAD hospital rates significantly increased after changing from cell culture cytotoxicity assay to a real-time PCR assay. One hundred ninety-nine hospitalized subjects were enrolled, and 101 fecal specimens were collected. C. difficile was detected in 18 subjects (18%), including 5 subjects (28%) with either definite or probable CDAD and 13 patients (72%) with asymptomatic C. difficile colonization.Conclusions.The majority of healthcare-associated diarrhea is not attributable to CDAD, and the prevalence of asymptomatic C. difficile colonization exceeds CDAD rates in healthcare facilities. PCR detection of asymptomatic C. difficile colonization among patients with non-CDAD diarrhea may be contributing to rising CDAD rates and a significant number of CDAD false positives. PCR may be useful for CDAD screening, but further study is needed to guide interpretation of PCR detection of C. difficile and the value of confirmatory tests. A gold standard CDAD diagnostic assay is needed.Infect Control Hosp Epidemiol 2014;35(6):667–673

Sign in / Sign up

Export Citation Format

Share Document