Optimization and validation of a real-time polymerase chain reaction protocol for the diagnosis of human brucellosis

Zeybek, Hasan; Açıkgöz, Ziya Cibali; Dal, Tuba; Durmaz, Rıza

doi:10.1007/s12223-019-00731-1

Cited by 8 publications

(6 citation statements)

References 39 publications

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“…These samples were used to estimate the diagnostic performance parameters of RT-PCR and conventional PCR targeting Brucella genomic DNA from serum samples of cows. Validation was performed according to [7] and AL-Ajlan et al [31]. On the contrary, these findings are in disagreement with the results reported by Tiwari et al [32] and Dal et al [33].…”

Section: Resultsmentioning

confidence: 70%

“…The diagnostic performance of conventional PCR and RT-PCR is affected by different factors such as the DNA extraction method, type of fluorogenic-labeled probe in case of RT-PCR, and the presence of foreign DNA and inhibitors in the samples [6]. Despite its high speed and diagnostic sensitivity (DSe) and specificity [7], the presence of inhibitors may decrease the sensitivity of PCR methods [8]. RT-PCR methods have been improved using TaqMan fluorogenic-labeled probes that exploit the 5′ nuclease activity of Taq DNA polymerase.…”

Section: Introductionmentioning

confidence: 99%

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Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera

et al. 2021

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Background and Aim: Different polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real- Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera. Materials and Methods: One hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard. Results: TaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%. Conclusion: A cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

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Section: Resultsmentioning

confidence: 70%

Section: Introductionmentioning

confidence: 99%

Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera

et al. 2021

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“…For comparison, the sensitivities of real-time or isothermal recombinase PCR are about 10 copies per reaction for Y. pestis (Qu et al, 2010), B. anthracis (Antwerpen et al, 2008;Bentahir et al, 2018), and Brucella spp. (Zeybek et al, 2019), which is equivalent to 2 × 10 3 -1 × 10 4 cells mL −1 in a 1-5 µL sample volume of template for each PCR, thereby enrichment of the target bacteria by culturing or extraction of nucleic acid is often required (Kane et al, 2019). Enrichment of the target bacteria, for example, by centrifugation, could be applied during sample preparation for the UPT-LF assay.…”

Section: Discussionmentioning

confidence: 99%

Calibration of an Upconverting Phosphor-Based Quantitative Immunochromatographic Assay for Detecting Yersinia pestis, Brucella spp., and Bacillus anthracis Spores

Zhang

Zhao

et al. 2020

Front. Cell. Infect. Microbiol.

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Yersinia pestis, Brucella spp., and Bacillus anthracis are pathogens that can cause infectious zoonotic diseases with high mortality rates. An upconverting phosphor-based quantitative immunochromatographic (UPT-LF) assay, a point-of-care testing method suitable for resource-limited areas, was calibrated to quantitatively detect pathogenic bacteria. The bacterial purity or activity were ensured via staining methods and growth curves, respectively. Growth assays showed that the classic plate-counting method underestimated bacterial numbers compared with the bacterial counting method recommended by the reference material of the National Institutes for Food and Drug Control, China. The detection results of the UPT-LF assay differed significantly between the bacterial cultures in liquid and solid media and between different strains. Accelerated stability assessments and freeze-thaw experiments showed that the stability of the corresponding antigens played an important role in calibrating the UPT-LF assay. In this study, a new calibration system was developed for quantitative immunochromatography for detecting pathogenic bacteria. The results demonstrated the necessity of calibration for standardizing point-of-care testing methods.

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“…Follow-up observations should be performed on those diagnosed with asymptomatic infection. Fortunately, previous studies had shown that polymerase chain reaction (PCR) testing has an important role in predicting the subsequent outcome of asymptomatic brucella infection [20][21][22][23] .…”

Section: Discussionmentioning

confidence: 99%

Screening household members of shepherds in brucellosis endemic areas in China and reporting follow-up results of asymptomatic infections

Hu¹,

Yang²,

Zhang³

et al. 2020

Preprint

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Background: Brucellosis is a zoonotic infectious disease caused by brucella, patients often show obvious clinical manifestation, however, many cases of asymptomatic brucella infection were reported. Previous scholars have described or screened the asymptomatic infection, but little attention has been paid to the results. This research focused on the short-term results in patients with asymptomatic brucella infectionMethods: 595 household members of shepherds in brucellosis endemic areas were included, all of them have questionnaires and laboratory tests. Based on inclusion and exclusion criteria for the cohort, 15 asymptomatic infections were included and followed-up for 18months.Results: Among 595 subjects, 34(5.7%) were asymptomatic infections, 460(77.3%) were healthy, 58patients (9.7%) were diagnosed as brucellosis, 13(2.2%) suspected cases, 19(3.2%) cured cases and 11(1.8%) unclear diagnosis. Among 15 asymptomatic infections, the median age was 34 [12, 50] years, there were 40%cases <18 years old and the male-female ratio was 1.5:1, 60% cases were farmers and herdsmen, 11(73.3%) cases had a history of possible exposure to brucella. Average follow-up time was 10.47 ± 8.47 months. A total of 7 asymptomatic infections developed into brucellosis, of which five patients turned in the first month of follow-up, one patient in the second month, and one minor case turned in the seventh month. Remaining asymptomatic infections showed negative outcomes after 7 months of follow-up, among them, SAT titer decreased in two cases, no changes in SAT titer and clinical manifestations in six cases.Conclusions: Continued exposure to brucella may be a major risk factor for asymptomatic infection turn to brucellosis.

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Optimization and validation of a real-time polymerase chain reaction protocol for the diagnosis of human brucellosis

Cited by 8 publications

References 39 publications

Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera

Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera

Calibration of an Upconverting Phosphor-Based Quantitative Immunochromatographic Assay for Detecting Yersinia pestis, Brucella spp., and Bacillus anthracis Spores

Screening household members of shepherds in brucellosis endemic areas in China and reporting follow-up results of asymptomatic infections

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