scholarly journals Comparison of Different PCR Methods for Detection of Brucella spp. in Human Blood Samples

2011 ◽  
Vol 60 (1) ◽  
pp. 27-33 ◽  
Author(s):  
HISHAM H. AL-AJLAN ◽  
ABDELNASSER S.S. IBRAHIM ◽  
ALI A. AL-SALAMAH
Keyword(s):  
Blood Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Brucella Melitensis ◽  
Blood Analysis ◽  
Clinical Samples ◽  
Conventional Pcr ◽  
Serum Samples ◽  
Culture Methods ◽  
Blood Specimens

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.

scholarly journals Diagnosis of canine leptospirosis: evaluation of two PCR assays in comparison with the microagglutination test

2019 ◽  
Vol 39 (4) ◽  
pp. 255-262
Author(s):  
Paula L. Martin ◽  
Nestor O. Stanchi ◽  
Bibiana F. Brihuega ◽  
Estela Bonzo ◽  
Lucía Galli ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Urine Samples ◽  
Clinical Samples ◽  
Conventional Pcr ◽  
Serum Samples ◽  
Presumptive Diagnosis ◽  
Microagglutination Test ◽  
Pcr Assays

ABSTRACT: Canine leptospirosis is definitely diagnosed by demonstrating seroconversion in paired serum samples from the acute and convalescent period by the microagglutination test (MAT). However, the application of a polymerase chain reaction (PCR) assay can provide earlier confirmation of suspected cases. The objective of this study was to evaluate two PCR assays used in diagnosis of human leptospirosis (lipL32 real-time PCR and rrs conventional PCR) in cultured microorganisms and experimentally contaminated samples (whole blood, serum, urine), and investigate their applicability in clinical samples from dogs with presumptive diagnosis of leptospirosis by using the MAT as a reference. The analytical sensitivity of the lipL32 real-time PCR was 1 genome equivalent per reaction, whereas that for the rrs conventional PCR was 10 genome equivalents per reaction. Both assays amplified the pathogenic strains but were negative when evaluating the DNA of other microorganisms that may be present in clinical samples. The lipL32 real-time PCR detected 100 bacteria/mL in whole blood samples, 1000 bacteria/mL in serum samples and 10 bacteria/mL in urine samples, whereas the rrs conventional PCR detected 1000 bacteria/mL in whole blood and serum samples and 100 bacteria/mL in urine samples. Seven out of the 51 samples from dogs with presumptive diagnosis of leptospirosis were considered as confirmed cases. ThelipL32 real-time PCR detected positive results in six of the seven confirmed cases, whereas the rrs conventional PCR detected four. The PCR assays evaluated proved to be useful diagnostic tools in the confirmation of canine leptospirosis when used together with the MAT.

scholarly journals Molecular Diagnosis of Invasive Aspergillosis and Detection of Azole Resistance by a Newly Commercialized PCR Kit

2017 ◽  
Vol 55 (11) ◽  
pp. 3210-3218 ◽  
Author(s):  
Eric Dannaoui ◽  
Frédéric Gabriel ◽  
Manuel Gaboyard ◽  
Gaëlle Lagardere ◽  
Lucile Audebert ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Azole Resistance ◽  
Rrna Gene ◽  
28S Rrna ◽  
Serum Samples ◽  
Content Type ◽  
28S Rrna Gene

ABSTRACTAspergillus fumigatusis the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance inA. fumigatusis worrisome. The aim of this study was to validate the new MycoGENIEA. fumigatusreal-time PCR kit and to evaluate its performance on clinical samples for the detection ofA. fumigatusand its azole resistance. This multiplex assay detects DNA from theA. fumigatusspecies complex by targeting the multicopy 28S rRNA gene and specific TR34and L98H mutations in the single-copy-numbercyp51Agene ofA. fumigatus. The specificity ofcyp51Amutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinicalA. fumigatusisolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for theAspergillus28S rRNA gene and 6 copies for thecyp51Agene harboring the TR34and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR34and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection ofA. fumigatusDNA and azole resistance due to TR34and L98H mutations in clinical samples.

Real time PCR assay for differentiation of Brucella abortus and Brucella melitensis

2017 ◽  
Author(s):  
Vinay Kumar ◽  
Sushila Maan ◽  
Aman Kumar ◽  
Kanisht Batra ◽  
Deepika Deepika ◽  
...  
Keyword(s):  
Real Time ◽  
Brucella Abortus ◽  
Real Time Pcr ◽  
Brucella Melitensis ◽  
Economic Losses ◽  
Conventional Pcr ◽  
Serological Tests ◽  
Detectable Difference ◽  
Pcr Assays ◽  
Respective Species

Brucellosis is one of the zoonotic diseases of major concern and can cause huge economic losses to livestock industry. Serological tests and bacterial isolation are considered as the gold standard assay for diagnosis of Brucella spp. but they are time-consuming, hazardous and lack specificity. To control and eradicate a disease, a confirmatory diagnostic method which is sensitive, quick and specific is the foremost requirement. Therefore in this study, we evaluated the performances of two newly designed TaqMan real-time PCR assays targeting the BruAB_0168 gene and BMEII0466 gene for Brucella abortus and Brucella melitensis (respectively). Both the assays were found to be highly specific in differentiation of respective species. Both the assays can detect as low as 0.02 fg of DNA and there was no detectable difference found in sensitivity of these two tests. R2 value and efficiency of these tests ranged from 0.992 - 0.998 and 100- 106%, respectively showing that these assays are highly efficient. Compared to conventional PCR assays these qPCR assays were 100 times higher sensitive. In conclusion, the present study showed that the developed real-time PCR assays are more sensitive, specific, have high reproducibility and repeatability and are faster than serological and conventional PCR methods for differentiation of Brucella abortus and Brucella melitensis.

scholarly journals Real-time PCR for detecting circulating dengue virus in the Guangdong Province of China in 2006

2008 ◽  
Vol 57 (12) ◽  
pp. 1547-1552 ◽  
Author(s):  
Zhijun Bai ◽  
Licheng Liu ◽  
Zeng Tu ◽  
Lisi Yao ◽  
Jianwei Liu ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
Serum Samples ◽  
Serological Tests ◽  
Wide Range ◽  
Pcr Assays

Dengue virus (DENV) causes a wide range of diseases in humans, from the acute febrile illness dengue fever (DF) to life-threatening dengue haemorrhagic fever/dengue shock syndrome. We developed four real-time quantitative PCR assays for each serotype of DENV based on computational analysis. These assays had high sensitivity and specificity without cross-reactivity for the four serotypes. To evaluate the performance of these assays in detecting and typing the virus in clinical samples, we analysed 64 serum samples from Guangdong during 2006. The results showed that 71 % of those samples were positive by the DEN-1 assay. The DENV assay results, in agreement with the serological tests and sequencing analysis, showed that the pathogen resulting in the DF explosion in Guangdong in 2006 belonged to DEN-1. Compared to the serological assays, the real-time PCR assays that we developed were much more sensitive in the 1–3 days after onset of the symptoms.

scholarly journals Diagnostic Performance of a Novel Multiplex PCR Assay for Candidemia among ICU Patients

2019 ◽  
Vol 5 (3) ◽  
pp. 86 ◽  
Author(s):  
Fuchs ◽  
Lass-Flörl ◽  
Posch
Keyword(s):  
Blood Culture ◽  
Diagnostic Accuracy ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Icu Patients ◽  
Blood Samples ◽  
Multiplex Pcr Assay ◽  
Whole Blood Samples

Candidemia poses a major threat to ICU patients and is routinely diagnosed by blood culture, which is known for its low sensitivity and long turnaround times. We compared the performance of a novel, Candida-specific multiplex real-time PCR assay (Fungiplex® Candida IVD Real-Time PCR Kit) with blood culture and another established diagnostic real-time PCR assay (LightCycler SeptiFast Test) with respect to Candida detection from whole blood samples. Clinical samples from 58 patients were analyzed by standard blood culture (BC) and simultaneously tested with the Fungiplex Candida PCR (FP) and the SeptiFast test (SF) for molecular detection of Candida spp. Compared to BC, the FP test showed high diagnostic power, with a sensitivity of 100% and a specificity of 94.1%. Overall diagnostic accuracy reached 94.6%. Using SF, we found a sensitivity of 60%, a specificity of 96.1%, and an overall diagnostic accuracy of 92.9%. The Fungiplex Candida PCR has shown good sensitivity and specificity on clinical samples of high-risk patients for direct detection of Candida species in whole blood samples. Together with conventional diagnostics (BC and antigen testing), this new multiplex PCR assay may contribute to a rapid and accurate diagnosis of candidiasis.

scholarly journals Development and Evaluation of Alternative Methods to Identify the Three Most Common Serotypes of Salmonella enterica Causing Clinical Infections in Kazakhstan

2021 ◽  
Vol 9 (11) ◽  
pp. 2319
Author(s):  
Sabyrkhan M. Barmak ◽  
Yuriy A. Sinyavskiy ◽  
Aidar B. Berdygaliev ◽  
Turegeldy Sh. Sharmanov ◽  
Irina S. Savitskaya ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Alternative Methods ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Screening Methods ◽  
Conventional Pcr ◽  
Diagnostic Efficacy ◽  
Microbial Cells

In this study, we aimed to compare the performance of conventional PCR and real-time PCR assays as screening methods for identification of three frequent, clinically significant Salmonella serovars in Kazakhstan. We determined the diagnostic efficacy of three molecular methods for detection of S. enterica subsp. enterica and typing S. Typhimurium, S. Enteritidis, and S. Virchow. A total of 137 clinical samples and 883 food samples were obtained in Almaty in 2018–2019. All tests showed high analytical specificity for detecting S. enterica and its corresponding serovariants (100%). The sensitivity of real-time PCR for each of the tested targets was 1–10 microbial cells and in conventional PCR 10–100 microbial cells. The trials with conventional PCR and real-time PCR had a diagnostic efficacy (DE) of 100% and 99.71%, respectively. The DE of real-time PCR and conventional PCR for detecting S. Enteritidis and S. Typhimurium was 99.90%, while the DE of conventional PCR and real-time PCR for detecting S. Virchow was 99.31% and 99.80%, respectively. The RAPD-PCR analysis of the genomic DNA of Salmonella enterica showed the genetic kinship of S. Enteritidis isolates, and the genetic heterogeneity of S. Typhimurium and S. Virchow isolates. Thus, the developed methods can be considered as alternatives to classical serotyping using antisera.

Rapid detection of Chlamydia/Chlamydophila group in samples collected from swine herds with and without reproductive disorders

2014 ◽  
Vol 17 (2) ◽  
pp. 367-369 ◽  
Author(s):  
K. Rypula ◽  
A. Kumala ◽  
P. Lis ◽  
K. Niemczuk ◽  
K. Płoneczka-Janeczko ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Complement Fixation Test ◽  
Complement Fixation ◽  
Fixation Test ◽  
Reproductive Disorders ◽  
Serum Samples ◽  
Swine Herds

Abstract The study was carried out in seven reproductive herds of pigs. In three of them reproductive disorders were observed. Three herds consisted of 10-50 and four consisted of 120-500 adult sows and they were called small and medium, respectively. Fifty-seven adult sows were randomly selected from herds. Serum samples were tested using the complement fixation test and swabs from both eyes and from the vaginal vestibule were examined using real-time PCR. All serum samples were negative. Infected sows were present in each of the study herds. In total, there were 28 positive samples (53%, 28/48) in real-time PCR in sows with reproductive disorders and 35 (53%, 35/66) in sows selected from herds without problems in reproduction. One isolate proved to be Chlamydophila pecorum, whereas all the remaining were Chamydia suis

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

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