Development and Use of Polymerase Chain Reaction for the Specific Detection of Salmonella Typhimurium in Stool and Food Samples

1999 ◽  
Vol 62 (10) ◽  
pp. 1103-1110 ◽  
Author(s):  
JER-SHENG LIN ◽  
HAU-YANG TSEN
Keyword(s):  
Polymerase Chain Reaction ◽  
Salmonella Typhimurium ◽  
Pcr Primers ◽  
Food Samples ◽  
Clinical Samples ◽  
Specific Detection ◽  
Chain Reaction ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Salmonella Serovars

Salmonella Typhimurium is one of the most important Salmonella serovars that may cause foodborne disease and human salmonellosis infection. Detection of this organism in the clinical samples of persons with gastroenteritis and the food samples associated with such persons may allow us to trace the cause of disease. Because malic acid dehydrogenase, an enzyme of the citric acid cycle, is common to organisms, the gene (mdh) coding for this enzyme was selected for the design of Salmonella Typhimurium-specific polymerase chain reaction (PCR) primers. By comparison of the mdh gene sequences of Salmonella Typhimurium and other Salmonella serotypes and of some isolates of other genera, two oligonucleotides were designed and used as PCR primers for the specific detection of Salmonella Typhimurium. The molecular weight of the PCR product was 261 bp as expected. Salmonella serovars other than Salmonella Typhimurium and isolates of other genera in the Enterobacteriaceae that is closely related to Salmonella did not generate any false-positive results. When this primer pair was used for the detection of Salmonella Typhimurium cells artificially inoculated into human stool specimens and food samples, such as milk and raw chicken meat, levels as low as 100 CFU per 0.1 g of stool specimen or per ml of milk or food homogenate could be detected if an 8- to 12-h preculture step using combined lactose-tetrathionate broth was performed prior to the PCR.

scholarly journals Specific detection ofSalmonella entericaserotype Enteritidis using the polymerase chain reaction

1996 ◽  
Vol 116 (2) ◽  
pp. 137-145 ◽  
Author(s):  
K. A. Lampel ◽  
S. P. Keasler ◽  
D. E. Hanes
Keyword(s):  
Polymerase Chain Reaction ◽  
Broth Culture ◽  
Virulence Plasmid ◽  
Specific Detection ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Single Base ◽  
Pcr Product ◽  
Base Mismatch ◽  
Polymerase Chain

SUMMARYAn assay was developed for the specific detection ofSalmonella entericaserotype Enteritidis, using a novel application of the polymerase chain reaction (PCR). This PCR assay is based on the mismatch amplification mutation assay, an allele-specific reaction, and can discriminate Enteritidis from all other salmonella. PCR primers were selected to amplify a 351-base pair (bp) DNA fragment from the salmonella plasmid virulence A (spvA) gene of Enteritidis. A single base difference at position 272 is present between the nucleotide sequence of thespvAgene of Enteritidis and other salmonellae. The downstream PCR primer, that encompasses position 272 of the EnteritidisspvAgene, was designed to contain a single base mismatch at the penultimate position, resulting in a l-base mismatch with Enteritidis and a 2-base mismatch with other salmonellae that harbour the virulence plasmid. The upstream primer was completely homologous with the region immediately 5′ to thespvAgene. When these primers were used and the annealing and extension reactions were performed at the same temperature, the PCR assay was specific for Enteritidis; no PCR product was detected for 40 other serotypes and 28 different genera examined. In pure culture, 120 colony forming units (c.f.u.) could be detected; a PCR product was observed from template derived from a 5 h enrichment broth culture of chicken seeded with 1 c.f.u. per gram of Enteritidis. This PCR assay is specific, reproducible, and less time consuming than the standard bacteriological methods used to detect Enteritidis.

Real-Time and Conventional Polymerase Chain Reaction Systems Based on the Metallo-Carboxypeptidase Inhibitor Gene for Specific Detection and Quantification of Potato and Tomato in Processed Food

2003 ◽  
Vol 66 (6) ◽  
pp. 1063-1070 ◽  
Author(s):  
MARTA HERNÁNDEZ ◽  
ALEJANDRO FERRANDO ◽  
TERESA ESTEVE ◽  
PERE PUIGDOMÈNECH ◽  
SALOMÉ PRAT ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Conventional Polymerase Chain Reaction ◽  
Food Samples ◽  
Specific Detection ◽  
Chain Reaction ◽  
Carboxypeptidase Inhibitor ◽  
Polymerase Chain ◽  
Detection And Quantification ◽  
Inhibitor Gene

In this paper, a method for the specific detection and quantification of potato and tomato DNA in food samples with the use of conventional and real-time polymerase chain reaction (PCR) is described. This method is adequate for use in food quality routine assays involving highly processed samples for which very tiny amounts of DNA are expected. Detection was achieved by amplifying a region of the metallo-carboxypeptidase inhibitor gene from either the potato (PCI) or the tomato (MCPI) and by using specific primers complementary to the propeptide regions of these inhibitors, which were found to differ for the potato and tomato proproteins. Conventional and real-time PCR systems were based on the same potato- or tomato specific primer pairs, and quantification was carried out with a TaqMan chemistry-based probe. The methods developed proved to be very specific and sensitive and highly reliable for the identification and quantification of DNA from both plant species. In addition, the construction of plasmids pPAT and pTOM, suitable for use as external calibration standards for the elaboration of comparative amplification profiles, is reported. Limits of detection and quantification with the use of these plasmid standards are given. Specificity and copy number conservation among different cultivars were analyzed, and the reliability of these systems was tested through their application to the analysis of commercial food samples including potato and/or tomato as components.

Triple-Helical Capture Assay for Quantification of Polymerase Chain Reaction Products

1992 ◽  
Vol 38 (5) ◽  
pp. 687-694 ◽  
Author(s):  
C P Vary
Keyword(s):  
Polymerase Chain Reaction ◽  
Fluorescence Analysis ◽  
Automated Analysis ◽  
Pcr Primers ◽  
Reaction Products ◽  
Chain Reaction ◽  
Capture Assay ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

Abstract This method for rapid, automated analysis of polymerase chain reaction (PCR) products makes use of PCR primers containing 5'-polypyrimidine sequences. Polypyrimidine-"headed" primers confer to the PCR product the ability to form triple helical complexes with a third polypyrimidine oligonucleotide. Third-strand oligonucleotides are modified to serve as either capture reagents or detection reagents for PCR products. Automated quantitative measurement of the PCR product is achieved by using latex bead-based fluorescence analysis. The use of triple-instead of double-helical interactions avoids the usual requirements of complex blocking reagents, time- and labor-intensive washing steps, and long times for color development. The method also provides rapid, sequence-specific capture and detection of PCR products without the need to denature the double-stranded PCR product. The assay is demonstrated with use of both PCR primer-derived and endogenous triple-helix-forming sequences resulting from PCR of several bacterial and viral target nucleic acids.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

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