Real-time PCR method for qualitative and quantitative detection of Lactobacillus sakei group species targeting novel markers based on bioinformatics analysis

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.

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Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x-70.6.1373 ◽

2007 ◽

Vol 70 (6) ◽

pp. 1373-1378 ◽

Cited By ~ 30

Author(s):

ANNA-CLARA RÖNNER ◽

HANS LINDMARK

Keyword(s):

Detection Limit ◽

Real Time ◽

Dna Extraction ◽

Campylobacter Jejuni ◽

Real Time Pcr ◽

Close Correlation ◽

Quantitative Detection ◽

Quantitative Real Time Pcr ◽

Direct Plating ◽

Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

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Development of a mreB-targeted real-time PCR method for the quantitative detection of Vibrio harveyi in seawater and biofilm from aquaculture systems

Aquaculture ◽

10.1016/j.aquaculture.2020.735337 ◽

2020 ◽

Vol 525 ◽

pp. 735337 ◽

Cited By ~ 1

Author(s):

Julia Mougin ◽

Roxane Roquigny ◽

Marie-Agnès Travers ◽

Thierry Grard ◽

Maryse Bonnin-Jusserand ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Harveyi ◽

Quantitative Detection ◽

Pcr Method

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Multiplex Fast Real-Time PCR for Quantitative Detection and Identification of cos- and pac-Type Streptococcus thermophilus Bacteriophages

Applied and Environmental Microbiology ◽

10.1128/aem.00295-08 ◽

2008 ◽

Vol 74 (15) ◽

pp. 4779-4781 ◽

Cited By ~ 23

Author(s):

Beatriz del Rio ◽

María Cruz Martín ◽

Noelia Martínez ◽

Alfonso H. Magadán ◽

Miguel A. Alvarez

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Industrial Process ◽

Streptococcus Thermophilus ◽

Quantitative Detection ◽

Detection And Identification ◽

Pcr Method

ABSTRACT The fermentation of milk by Streptococcus thermophilus is a widespread industrial process that is susceptible to bacteriophage attack. In this work, a preventive fast real-time PCR method for the detection, quantification, and identification of types of S. thermophilus phages in 30 min is described.

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Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.02852-05 ◽

2006 ◽

Vol 72 (9) ◽

pp. 6040-6048 ◽

Cited By ~ 42

Author(s):

Belï¿½n Martï¿½n ◽

Anna Jofrï¿½ ◽

Margarita Garriga ◽

Maria Pla ◽

Teresa Aymerich

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fluorescence Probe ◽

Plate Count ◽

23S Rrna ◽

Quantitative Detection ◽

Lactobacillus Sakei ◽

Simple Method ◽

Chelex 100 ◽

Fermented Sausages

ABSTRACT A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.

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A novel Alu-based real-time PCR method for the quantitative detection of plasma circulating cell-free DNA: Sensitivity and specificity for the diagnosis of myocardial infarction

International Journal of Molecular Medicine ◽

10.3892/ijmm.2014.1991 ◽

2014 ◽

Vol 35 (1) ◽

pp. 72-80 ◽

Cited By ~ 13

Author(s):

XIAOLI LOU ◽

YANQIANG HOU ◽

DONGYU LIANG ◽

LIANG PENG ◽

HONGWEI CHEN ◽

...

Keyword(s):

Myocardial Infarction ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Quantitative Detection ◽

Cell Free Dna ◽

Free Dna ◽

Pcr Method ◽

Circulating Cell Free Dna

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Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy

Canadian Journal of Microbiology ◽

10.1139/cjm-2016-0482 ◽

2017 ◽

Vol 63 (4) ◽

pp. 296-302 ◽

Cited By ~ 8

Author(s):

Massimiliano Bergallo ◽

Ilaria Galliano ◽

Paola Montanari ◽

Martina Rosa Brusin ◽

Serena Finotti ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Acute Gastroenteritis ◽

Taqman Assay ◽

Quantitative Detection ◽

Common Disease ◽

Median Viral Load ◽

Significant Difference ◽

Pcr Method ◽

Children Under 5

Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log10) ± 7.1(log10) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

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