scholarly journals The Sensitivity of the Real-time PCR and Nested-PCR for Detection of Coxiella burnetii in Milk Samples

2017 ◽  
Vol 4 (2) ◽  
pp. 11
Author(s):  
Mojtaba Bonyadian ◽  
Hamdollah Moshtaghi ◽  
Hamidreza Kazemeini
Keyword(s):  
Real Time ◽  
Coxiella Burnetii ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Pcr Assay ◽  
Milk Samples ◽  
Bulk Milk ◽  
Significant Difference ◽  
Pcr Assays ◽  
Nested Pcr Assays

Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. In humans serology is the gold standard for diagnosis but is inadequate for early case detection, so real-time PCR and nested-PCR assays were developed in this study to measure amounts of C.burnetii shed in milk. Our study was to assess the sensitivity of the realtime PCR and nested-PCR for detection of Coxiella burnetii in bovine bulk milk samples from dairy herds in 3 provinces (Chaharmahal and Bakhtiari , Isfahan and Yazd) of Iran. In the present study, 300 bulk milk samples from 89 dairy cattle herds were tested for C. burnetii using real-time PCR and nested-PCR assays. The animals which their milk samples collected for this study were clinically healthy. In total, 74 of 300 (24.7%) cow milk samples were positive in real-time PCR assay and 26 of 300 (8.7%) samples were positive in nested-PCR assay. McNemar test shows a significant difference in detection of C. burnetii between real-time PCR and nested-PCR. Also the results of this study indicate those clinically healthy dairy cows are important sources of C. burnetii infection in Iran.

scholarly journals PP-040 Real-time PCR assay for identification of Brucella abortus in bulk milk samples in Iran

2010 ◽  
Vol 14 ◽  
pp. S36-S37 ◽  
Author(s):  
S. Moshkelani ◽  
M. Javaheri Koupaei ◽  
S. Rabiei ◽  
A. Doosti
Keyword(s):  
Real Time ◽  
Brucella Abortus ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Milk Samples ◽  
Bulk Milk

scholarly journals A Retrospective Study about the Impact of Switching from Nested PCR to Multiplex Real-Time PCR on the Distribution of the Human Papillomavirus (HPV) Genotypes

Medicina ◽  
2019 ◽  
Vol 55 (8) ◽  
pp. 418 ◽  
Author(s):  
Prete ◽  
Ronga ◽  
Addati ◽  
Magrone ◽  
Abbasciano ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Diagnostic Methods ◽  
Pcr Assay ◽  
Hpv Prevalence ◽  
Significant Difference ◽  
Hpv Genotypes ◽  
The Impact

Background and objectives: Human papillomavirus (HPV) is the most prevalent etiological agent of viral sexually-transmitted infection. This study retrospectively evaluated the impact of a switch to a real-time PCR assay in the HPV prevalence and genotypes distribution by a quasi-experimental before-and-after approach. Materials and Methods: In total, 1742 samples collected from 1433 patients were analyzed at the UOC Microbiology and Virology of Policlinico of Bari, Italy. HPV DNA detection was performed using initially nested PCR and subsequently multiplex real-time PCR assay. Results: Statistically significant difference in HPV overall prevalence after the introduction of the real-time assay was not detected (48.97% vs. 50.62%). According to different extraction-DNA amplification methods, differences were observed in the prevalence rates of HPV-45, 68, 40, 42, and 43. The lowest prevalence for HPV-45 was observed in the Magna Pure-Real Time PCR group, while HPV-68, 40, 42, and 43 were less observed in the Qiagen-Real Time PCR group. After, a multivariate logistic regression, an increase in the prevalence of HPV-42 (aOR: 4.08, 95% CI: 1.71–9.73) was associated with the multiplex real-time PCR assay. Conclusions: Although this study is a not a direct comparison between two diagnostic methods because it has a sequential structure, it serves to verify the impact of a new molecular assay on HPV distribution. Moreover, the stability of HPV prevalence over time suggests that the population composition and the behavioral variables did not likely change during the observation period. Our study proposes that the introduction of a molecular test for HPV detection may be related to changes of HPV genotypes distribution.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

Multiplex, Construct-Specific, and Real-Time PCR-Based Analytical Methods for Bt Rice with cry1Ac Gene

2012 ◽  
Vol 95 (1) ◽  
pp. 186-194 ◽  
Author(s):  
Gurinder Jit Randhawa ◽  
Monika Singh
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Analytical Methods ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Pcr Assay ◽  
Cry1ac Gene ◽  
Camv 35S ◽  
Bt Rice ◽  
Pcr Assays

Abstract Qualitative and quantitative analytical methods based on PCR for Bacillus thuringiensis (Bt) rice hybrid, namely, MRP 5401 Bt expressing a modified version of the Bt cry1Ac gene, are reported here. Multiplex PCR assays were developed to target the cry1Ac transgene, Cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptII) marker gene, and an endogenous α-tubulin (TubA) gene in Bt rice. The 3.178 kb region of inserted gene construct comprising the region of the CaMV 35S promoter and cry1Ac gene was amplified, and the construct integrity was confirmed by the nested PCR. The LOD for cry1Ac gene-specific simplex PCR was 0.01%, as established using Bt rice DNA dilutions with 100, 10, 1.0, 0.1, 0.05, 0.01, and 0.001% genetically modified trait. A real-time PCR assay was also developed to quantify the cry1Ac gene. The method performance of the reported real-time PCR assay was in line with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with LOD and LOQ values of 0.05%. The reliable PCR assays prior to commercial release of Bt rice would facilitate efficient regulatory compliance for identification of genetic trait, labeling requirements, and effective risk assessment and management. They could also address consumers' concerns and legal disputes that may arise.

Evaluation of a Real-Time PCR Assay to Detect Coxiella burnetii

2006 ◽  
Vol 1078 (1) ◽  
pp. 563-565 ◽  
Author(s):  
S. R KLEE ◽  
H. ELLERBROK ◽  
J. TYCZKA ◽  
T. FRANZ ◽  
B. APPEL
Keyword(s):  
Real Time ◽  
Coxiella Burnetii ◽  
Real Time Pcr ◽  
Pcr Assay

Diagnosis of intramammary infection in samples yielding negative results or minor pathogens in conventional bacterial culturing

2010 ◽  
Vol 78 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Ricardo Bexiga ◽  
Mikko T Koskinen ◽  
Jani Holopainen ◽  
Carla Carneiro ◽  
Helena Pereira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Gram Positive ◽  
Milk Samples ◽  
Negative Results ◽  
Streptococcus Uberis ◽  
Cell Counts ◽  
Significant Difference ◽  
Culture Negative

Up to half of quarter milk samples submitted for mastitis diagnosis are culture-negative results or lead to identification of coagulase-negative staphylococci or Corynebacterium bovis in conventional culturing, the so-called minor pathogens. The interpretation and usefulness of these results in terms of udder and animal health management is limited, even though the amount of resources spent is relatively high. This work aimed to test two methods of analysis of milk samples with the goal of increasing detection of intramammary pathogens. In the first study, 783 milk samples were processed in duplicate: before and after freezing at −20°C for 24 h, using standard bacteriological techniques. There was a significant difference between the two methods with samples frozen for 24 h yielding significantly fewer Gram-positive catalase-positive cocci, Gram-negative bacilli, Gram-positive bacilli and significantly more samples leading to no growth, than samples before freezing. The number of samples yielding Gram-positive catalase-negative cocci was not significantly affected by freezing. In the second study, a real-time PCR-based test was performed on milk samples with an individual quarter somatic cell count above 500 000 cells/ml that were either negative (n=51 samples) or that led to the isolation of minor pathogens in culturing: Corynebacterium bovis (n=79 samples) or non-aureus staphylococci (NAS, n=32). A mastitis pathogen, beyond the result obtained with standard bacteriology, was detected on 47% of the no-growth samples, on 35% of the samples from which C. bovis had been isolated and on 25% of the samples from which NAS had been isolated. The most commonly detected major pathogen was Escherichia coli, followed by Streptococcus uberis, Arcanobacterium pyogenes/Peptoniphilus indolicus and Streptococcus dysgalactiae. These results suggest that simply freezing milk samples for 24 h does not increase the detection of intramammary bacteria in milk samples and therefore should not be recommended. However, use of the real-time PCR-based test may be useful in diagnosing intramammary infections when milk samples with high somatic cell counts are culture-negative or when culturing results in the detection of minor pathogens.

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