Evaluation of a Polymerase Chain Reaction–Based Test for Detecting Salmonella spp. in Food Samples: Probelia Salmonella spp.

1999 ◽  
Vol 62 (12) ◽  
pp. 1387-1393 ◽  
Author(s):  
PATRICK FACH ◽  
FRANÇOISE DILASSER ◽  
JOËL GROUT ◽  
JOCELYNE TACHE
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Collaborative Study ◽  
Reference Method ◽  
Food Samples ◽  
Complete Agreement ◽  
Chain Reaction ◽  
Salmonella Spp ◽  
Sandwich Hybridization ◽  
Polymerase Chain

A commercially available polymerase chain reaction (PCR) kit was evaluated for the detection of Salmonella spp. in food samples. The test combines PCR amplification and sandwich hybridization of the amplified DNA in microtiter plates. The sensitivity and specificity was evaluated with 52 Salmonella strains and 51 non-Salmonella strains and showed that the test was entirely reliable. The threshold sensitivity was 102 CFU/ml. The limit of detection of dead cells that determines the minimum detection level of dead cells in food samples was superior to 106 CFU/25 g, a level rarely achieved in naturally contaminated samples. After an 18-h pre-enrichment step, the test could detect viable Salmonella in artificially contaminated food samples, even for the lower contamination level (3 CFU/25 g). There was complete agreement between the PCR test and the ISO 6579 bacteriological reference method with artificially contaminated samples. Regarding the accuracy of the results obtained from 253 naturally or noncontaminated foods and from 32 artificially contaminated foods, the agreement percentage was 99.6%. The fidelity of the technique was evaluated in a collaborative study with eight European laboratories and showed a correlation of 98.4%.

Combination of Immunomagnetic Separation and Polymerase Chain Reaction for the Simultaneous Detection of Listeria monocytogenes and Salmonella spp. in Food Samples

2001 ◽  
Vol 64 (11) ◽  
pp. 1744-1750 ◽  
Author(s):  
HSIEN-YEE HSIH ◽  
HAU-YANG TSEN
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Simultaneous Detection ◽  
Meat Products ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Chain Reaction ◽  
Salmonella Spp ◽  
Pcr Method ◽  
Polymerase Chain

A method that combined the immunomagnetic separation (IMS) technique and the multiplex polymerase chain reaction (PCR) method (i.e., the IMS-mPCR method) was developed for simultaneous detection of Listeria monocytogenes and Salmonella spp. in food samples. When only the multiplex PCR method was used, it was found that if cell numbers of each of the two target organisms (L. monocytogenes and Salmonella spp.) were above the detection limit, but differed by more than 2 logs—e.g., n × 107 to n × 104 or n × 106 to n × 103—the organism presenting the lower numbers might go undetected. Following the enrichment step with universal preenrichment (UP) broth, if an IMS method using equal quantities of anti-Listeria and anti-Salmonella immunomagnetic beads was performed prior to PCR, both pathogens could be detected unambiguously. Such results could be obtained for target organisms in food samples, such as milk, dairy, and meat products, if similar enrichment and IMS steps were performed prior to PCR.

scholarly journals Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases

2021 ◽  
Author(s):  
Jing Xu ◽  
Timothy Kirtek ◽  
Yan Xu ◽  
Hui Zheng ◽  
Huiyu Yao ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
False Negative ◽  
Accurate Method ◽  
Viral Envelope ◽  
Chain Reaction ◽  
Borderline Cases ◽  
Droplet Pcr ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Abstract Objectives The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR—in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. Methods We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer’s specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription–polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. Results The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. Conclusions The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

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.

Polymerase Chain Reaction Detection of Salmonella spp. in Fecal Samples of Pet Birds

2008 ◽  
Vol 3 (1) ◽  
pp. e29-e29
Author(s):  
B. Sareyyüpoğlu ◽  
A Çelik Ok ◽  
Z. Cantekin ◽  
H. Yardimci ◽  
M. Akan ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Detection ◽  
Chain Reaction ◽  
Salmonella Spp ◽  
Fecal Samples ◽  
Pet Birds ◽  
Polymerase Chain

Comparison of Droplet Digital Polymerase Chain Reaction (ddPCR) and Real-Time Quantitative Polymerase Chain Reaction (qPCR) in Detecting Neonatal Invasive Fungal Infections

2021 ◽  
Vol 11 (3) ◽  
pp. 373-379
Author(s):  
Huitao Li ◽  
Xueyu Chen ◽  
Xiaomei Qiu ◽  
Weimin Huang ◽  
Chuanzhong Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Fungal Isolation ◽  
Blood Samples ◽  
Fungal Strains ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Invasive fungal infection (IFI) is the leading cause of death in neonatal patients, yet the diagnosis of IFI remains a major challenge. At present, most IFI laboratory diagnostic methods are based on classical, but limited, methods such as fungal isolation and culture and histopathological examination. Recently, quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) technology have been adopted to quantify nucleic-acid identification. In this study, we established qPCR and ddPCR assays for IFI diagnosis and quantification. qPCR and ddPCR were carried out using identical primers and probe for the amplification of 18S rRNA. Assay results for three fungal strains were positive, whereas ten non-fungal strains had negative results, indicating 100% specificity for both ddPCR and qPCR methods. Genomic DNA of Candida albicans was tested after a serial dilution to compare the sensitivity of the two PCR methods. The limit of detection of ddPCR was 3.2 copies/L, which was a ten-fold increase compared with that of the qPCR method (32 copies/L). Blood samples from 127 patients with high-risk factors and clinical symptoms for IFI were collected from a NICU in Shenzhen, China, and analyzed using qPCR and ddPCR. Thirty-four blood samples from neonates had a proven or probable diagnosis of IFI, and 25 of these were positive by qPCR, whereas 30 were positive by ddPCR. Among the 93 blood samples from neonates who had a possible IFI or no IFI, 24 were positive using qPCR, and 7 were positive using ddPCR. In conclusion, ddPCR is a rapid and accurate pan-fungal detection method and provides a promising prospect for IFI clinical screening.

scholarly journals Rapid Rh D genotyping by polymerase chain reaction-based amplification of DNA

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2975-2980 ◽  
Author(s):  
S Simsek ◽  
BH Faas ◽  
PM Bleeker ◽  
MA Overbeeke ◽  
HT Cuijpers ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Group ◽  
False Positive ◽  
Complete Agreement ◽  
Blood Group System ◽  
Chain Reaction ◽  
Group System ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Rh Blood Group

Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3′ noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.

scholarly journals Validation of Droplet Digital Polymerase Chain Reaction for Salmonella spp. Quantification

2020 ◽  
Vol 11 ◽  
Author(s):  
Carolina Villamil ◽  
Martha Nancy Calderon ◽  
Maria Mercedes Arias ◽  
John Emerson Leguizamon
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Salmonella Spp ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction
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