scholarly journals Development of a Genoserotyping Method for Salmonella Infantis Detection on the Basis of Pangenome Analysis

2020 ◽  
Vol 9 (1) ◽  
pp. 67
Author(s):  
Seung-Min Yang ◽  
Jiwon Baek ◽  
Eiseul Kim ◽  
Hyeon-Be Kim ◽  
Seyoung Ko ◽  
...  
Keyword(s):  
Public Health ◽  
Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Cost Effective ◽  
The Other ◽  
Gene Marker ◽  
Diagnostic Assay ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Substantial Economic Burden

In recent years, Salmonella Infantis has become a predominant serovariant in clinical and poultry isolates, thereby imposing a substantial economic burden on both public health and the livestock industry. With the aim of coping with the steep increase in serovar Infantis prevalence, a polymerase chain reaction (PCR)-based rapid and accurate diagnostic assay was developed in this study through pangenome profiling of 60 Salmonella serovars. A gene marker, SIN_02055, was identified, which is present in the S. Infantis genome but not in the pangenome of the other serovars. Primers specific to SIN_02055 were used to accurately detect serovar Infantis, and to successfully differentiate Infantis from the other 59 serovars in real-time PCR with a R2 of 0.999 and an efficiency of 95.76%. The developed method was applied to 54 Salmonella strains belonging to eight dominant serovars, and distinguished Infantis from the other seven serovars with an accuracy of 100%. The diagnostic primer set also did not show false positive amplification with 32 strains from eight non-Salmonella bacterial species. This cost-effective and rapid method can be considered an alternative to the classic serotyping using antisera.

scholarly journals Development of Quantitative Real-Time Polymerase Chain Reaction for Detection of and Discrimination between Erysipelothrix Rhusiopathiae and Other Erysipelothrix Species

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Practical Applications ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

scholarly journals Identification of Neisseria gonorrhoeae isolates with a recombinant porA gene in Scotland, United Kingdom, 2010 to 2011

2012 ◽  
Vol 17 (9) ◽  
Author(s):  
K Eastick ◽  
A Winter ◽  
S Jamdar
Keyword(s):  
Polymerase Chain Reaction ◽  
United Kingdom ◽  
Neisseria Gonorrhoeae ◽  
Sequence Type ◽  
The Other ◽  
Chain Reaction ◽  
Polymerase Chain

Three isolates of Neisseria gonorrhoeae have been identified in Scotland in 2010 and 2011, which lack sequences in the porA pseudogene commonly used as the target for confirmatory gonorrhoea polymerase chain reaction assays. Two isolates were clustered temporally and geographically and have the same sequence type and porA sequence. A similar strain was reported in Australia during early 2011. The other Scottish isolate was identified separately and is different in sequence type and porA sequence.

scholarly journals High-Resolution Melting-Based Quantitative Analysis of RASSF1 Methylation in Breast Cancer

Medicina ◽  
2013 ◽  
Vol 49 (2) ◽  
pp. 14 ◽  
Author(s):  
Kristina Stuopelytė ◽  
Kristina Daniūnaitė ◽  
Aida Laurinavičienė ◽  
Valerijus Ostapenko ◽  
Sonata Jarmalaitė
Keyword(s):  
Breast Cancer ◽  
Polymerase Chain Reaction ◽  
High Resolution ◽  
Quantitative Methods ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Chain Reaction ◽  
Specific Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Breast Tissues

Background and Objective. Breast cancer is the leading cause of death from cancer among women worldwide. The aberrant promoter methylation of tumor suppressor genes is a typical epigenetic alteration for breast cancer and can be detected in early carcinogenesis. High-throughput and cost-effective methods are needed for the early and sensitive detection of epigenetic changes in clinical material. The main purpose of our study was to optimize a high-resolution melting (HRM) assay for the reliable and quantitative assessment of RASSF1 gene methylation, which is considered one of the earliest epigenetic alterations in breast cancer. Material and Methods. A total of 76 breast carcinomas and 10 noncancerous breast tissues were studied by means of HRM and compared with the results obtained by means of quantitative methylation-specific polymerase chain reaction (QMSP) and methylation-specific polymerase chain reaction (MSP). Results. Both quantitative methods, HRM and QMSP, showed a similar specificity and sensitivity for the detection of RASSF1 methylation in breast cancer (about 80% and 70%, respectively). In breast cancer, the mean methylation intensity of RASSF1 was 42.5% and 48.6% according to HRM and QMSP, respectively. Both methods detected low levels of methylation (less than 5%) in noncancerous breast tissues. In comparison with quantitative methods, MSP showed a lower sensitivity (70%), but a higher specificity (80%) for the detection of RASSF1 methylation in breast cancer. Conclusions. HRM is as a simple, cost-effective method for the reliable high-throughput quantification of DNA methylation in clinical material.

Identification and diagnosis of Leishmania mexicana complex isolates by polymerase chain reaction

Parasitology ◽  
1994 ◽  
Vol 109 (4) ◽  
pp. 423-433 ◽  
Author(s):  
S. Eresh ◽  
S. M. McCallum ◽  
D. C. Barker
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
The Other ◽  
South American ◽  
Kinetoplast Dna ◽  
Leishmania Mexicana ◽  
Chain Reaction ◽  
Potential Host ◽  
Oligonucleotide Primers ◽  
Polymerase Chain

SUMMARYFollowing cloning of Leishmania (L.) amazonensis kinetoplast DNA two recombinant clones were identified: one specific for L. (L.) amazonensis and the other specific for L. (L.) amazonensis and closely related isolates. DNA sequences from these clones were compared with those of other kinetoplastids and oligonucleotide primers were designed to be used in the polymerase chain reaction. A pair of these primers has been shown not only to be highly specific for L. mexicana complex isolates but can also be used to distinguish between L. (L.) mexicana and L. (L.) amazonensis isolates. These primers have been tested with water-lysed cultures, crude DNA extracts from human patients, potential host reservoirs, sandfly vectors and with cell pellets after isoenzyme characterization. The results of these tests indicate that the primers can be used specifically in the presence of excess host DNA originating from the majority of South American countries.

Molecular typing of Lactobacillus brevis isolates from Korean food using repetitive element-polymerase chain reaction

2018 ◽  
Vol 24 (4) ◽  
pp. 341-350 ◽  
Author(s):  
Jasmine Kaur ◽  
Anshul Sharma ◽  
Sulhee Lee ◽  
Young-Seo Park
Keyword(s):  
Polymerase Chain Reaction ◽  
Repetitive Element ◽  
Bacterial Species ◽  
Large Family ◽  
Lactobacillus Brevis ◽  
Polymerase Chain Reaction Method ◽  
Fermented Foods ◽  
Chain Reaction ◽  
Easy Method ◽  
Polymerase Chain

Lactobacillus brevis is a part of a large family of lactic acid bacteria that are present in cheese, sauerkraut, sourdough, silage, cow manure, feces, and the intestinal tract of humans and rats. It finds its use in food fermentation, and so is considered a “generally regarded as safe” organism. L. brevis strains are extensively used as probiotics and hence, there is a need for identifying and characterizing these strains. For identification and discrimination of the bacterial species at the subspecific level, repetitive element-polymerase chain reaction method is a reliable genomic fingerprinting tool. The objective of the present study was to characterize 13 strains of L. brevis isolated from various fermented foods using repetitive element-polymerase chain reaction. Repetitive element-polymerase chain reaction was performed using three primer sets, REP, Enterobacterial Repetitive Intergenic Consensus (ERIC), and (GTG)5, which produced different fingerprinting patterns that enable us to distinguish between the closely related strains. Fingerprinting patterns generated band range in between 150 and 5000 bp with REP, 200–7500 bp with ERIC, and 250–2000 bp with (GTG)5 primers, respectively. The Jaccard’s dissimilarity matrices were used to obtain dendrograms by the unweighted neighbor-joining method using genetic dissimilarities based on repetitive element-polymerase chain reaction fingerprinting data. Repetitive element-polymerase chain reaction proved to be a rapid and easy method that can produce reliable results in L. brevis species.

Predictive Value and Cost-Effectiveness Analysis of a Rapid Polymerase Chain Reaction for Preoperative Detection of Nasal Carriage of Staphylococcus aureus

2003 ◽  
Vol 24 (5) ◽  
pp. 327-333 ◽  
Author(s):  
Nabin K. Shrestha ◽  
Kenneth M. Shermock ◽  
Steven M. Gordon ◽  
Marion J. Tuohy ◽  
Deborah A. Wilson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Cost Effectiveness ◽  
Cost Effective ◽  
Nasal Carriage ◽  
Initial Therapy ◽  
Cost Effectiveness Analysis ◽  
Chain Reaction ◽  
Effectiveness Analysis ◽  
Polymerase Chain

AbstractObjective:To determine the accuracy and cost-effectiveness of a polymerase chain reaction (PCR) for detecting nasal carriage of Staphylococcus aureus directly from clinical specimens.Cross-Sectional Study:This occurred in a tertiary-care hospital in Cleveland, Ohio, and included 239 consecutive patients who were scheduled for a cardiothoracic surgical procedure. Conventional cultures and a PCR for S. aureus from nasal swabs were used as measurements.Cost-Effectiveness Analysis:Data sources were market prices and Bureau of Labor Statistics. The time horizon was the maximum period for availability of culture results (3 days). Interventions included universal mupirocin therapy without testing; initial therapy, with termination if PCR negative (treat-PCR); initial therapy, with termination if culture negative (treat-culture); treat PCR-positive carriers (PCR-guided treatment); and treat culture-positive carriers (culture-guided treatment). The perspective was institutional and costs and the length of time to treatment were outcome measures.Results:Sixty-seven (28%) of the 239 swabs grew S. aureus. Rapid PCR was 97.0% sensitive and 97.1% specific for the detection of S. aureus. For populations with prevalences of nasal S. aureus carriage of up to 50%, the PCR assay had negative predictive values of greater than 97%. PCR-guided treatment had the lowest incremental cost-effectiveness ratio ($1.93 per additional day compared with the culture strategy). Among immediate treatment strategies, treat-PCR was most cost-effective. The universal therapy strategy cost $38.19 more per additional day gained with carrier identification compared with the PCR strategy.Conclusion:Rapid real-time PCR is an accurate, rapid, and cost-effective method for identifying S. aureus carriers for preoperative intervention.

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