scholarly journals Simplified Method of Detection of Dialister invisus and Olsenella uli in Oral Cavity Samples by Polymerase Chain Reaction

2017 ◽  
Vol 8 (1-2) ◽  
pp. 47-52 ◽  
Author(s):  
Manohar S. Kugaji ◽  
Kishore G. Bhat ◽  
Vinayak M. Joshi ◽  
Madhu Pujar ◽  
Pratik T. Mavani
Keyword(s):  
Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Universal Primers ◽  
Sample Collection ◽  
Chain Reaction ◽  
Infection Group ◽  
Endodontic Infection ◽  
Predominant Component ◽  
Endodontic Infections ◽  
Polymerase Chain

Aims and Objectives: The oral microbial flora is highly complex and diverse with obligate anaerobic bacteria as the predominant component. Most of these are not yet cultivated/difficult to cultivate due to technical limitations. In this study, we aim to detect two novel oral bacterial species Dialister invisus and Olsenella uli by simplified and economical procedure of polymerase chain reaction (PCR) and study their association with primary and persistent endodontic infections. Material and Methods: The study involved 60 patients that included 30 patients of primary endodontic infections and 30 with persistent endodontic infections. The sample collection from the root canal was performed by universally accepted protocol by using sterile paper points. The deoxyribonucleic acid (DNA) extraction was done, followed by PCR with species specific primers. We made several changes to the protocol mentioned by original authors. We adopted a one-step protocol for amplification of bacterial DNA, omitting the 16SrDNA amplification step with universal primers. Results: It was seen that 7 (23.3 %) samples in primary endodontic infection group and 24 (80 %) samples in persistent endodontic infection group were positive for D. invisus. On the other hand, 11 (36.6 %) patients of primary endodontic infection showed positivity for O. uli in comparison to 9 (30 %) of persistent endodontic infection. Conclusion: The results from the present study showed efficient amplification of both O. uli and D. invisus in a single-step PCR. Hence, we conclude that the modified protocol used here with taq polymerase enzyme offers a faster and cheaper alternative to nested PCR without compromising the quality of amplification process.

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 Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis

2017 ◽  
Vol 69 (6) ◽  
pp. 1443-1450 ◽  
Author(s):  
M.P. Campos ◽  
M.F. Madeira ◽  
D.A. Silva ◽  
M.S. Solcà ◽  
O.M. Espíndola ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Healthy Skin ◽  
Sample Collection ◽  
Chain Reaction ◽  
Dna Recovery ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Polymerase Chain ◽  
Commercial Kits

ABSTRACT The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery

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 Psittacine beak and feather disease virus in budgerigars and ring-neck parakeets in South Africa

2004 ◽  
Vol 71 (1) ◽  
Author(s):  
J. Albertyn ◽  
K.M. Tajbhai ◽  
R.R. Bragg
Keyword(s):  
South Africa ◽  
Polymerase Chain Reaction ◽  
Genetic Variation ◽  
Disease Virus ◽  
Common Disease ◽  
Sample Collection ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Feather Disease Virus ◽  
Viral Isolates

Psittacine beak and feather disease (PBFD) is a common disease of the psittacine species and is caused by the psittacine beak and feather disease virus (PBFDV). In this study the occurrence of the disease in ring-neck parakeets and budgerigars in South Africa suffering from feathering problems, using polymerase chain reaction as a diagnostic test was investigated. The genetic variation between viral isolates was also studied. Results indicate that PBFDV can be attributed to being the cause of feathering problems in some of the ring-neck parakeets and budgerigars in South Africa. Genetic variation of isolates occurs between species and individuals. A cheap and easy to use method of blood sample collection on filter paper for diagnostic purposes was also evaluated. It proved to be less stressful to the birds and did not inhibit further processes.

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.

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