Combined nucleic acid assays for diagnosis of A19 vaccine‐caused human brucellosis

Liu, Baoshan; Ye, Yin-Bo; Zhai, Jing; Zhang, Yi; Yang, Jun J.; Dai, Cheng; Chi, Ma; Donghai, Yu; Yang, Baofeng; Rongnian, Zhu; Feng, Sheng; Zhang, Jun; Han, Xiaohu; Chen, Zeliang

doi:10.1111/tbed.13685

Cited by 13 publications

(7 citation statements)

References 18 publications

(24 reference statements)

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“…However, under experimental conditions, it has been shown that some Brucella species could invade red blood cells and settle in the cytoplasm without replicating, establishing persistent bacteremia with different bacterial loads (Vitry et al, 2014;Gwida et al, 2016). In humans, a study of three patients infected in the recent Brucellosis outbreak in China con rmed that Brucella persists in peripheral blood (Baoshan et al, 2020). In this study, a high recovery of Brucella-DNA from whole bovine blood was possible.…”

Section: Discussionmentioning

confidence: 67%

“…Improvements in PCR methods such as real-time PCR (qPCR), have made it possible to detect Brucella spp. from clinical samples of human (Queipo-Ortuño et al, 1997; Baoshan et al, 2020 ), and animal (Wareth et al, 2015;Gwida et al, 2016;Selim et al, 2019), even when there is a low number of organisms (Hull et al, 2018), combining speed, sensitivity, high speci city (with use of probe), laboratory safety, and low risk of cross-contamination or amplicon contamination in the bloodstream (Corbel, 2006). The qPCR technique's application may support the drawbacks of the indirect methods and the low sensitivity of the microbial isolation technique.…”

Section: Introductionmentioning

confidence: 99%

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Epidemiology Aspects of Brucella-DNA Detection Using Real-time PCR from Bovine Whole Blood Samples in Colombia

Ramírez¹,

Santos²,

Paulino³

et al. 2021

Preprint

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A cross-sectional study was conducted in Colombia to recover Brucella spp. DNA from bovine whole-blood samples through probe-based real-time PCR (Probe-qPCR) and by an SNP-based assay, differentiating vaccine strains from field strains. The associated factors for the presence of Brucella-DNA were reported and evaluated using logistical regression models. A total of 656 random cows from 40 herds were selected. Template DNA was obtained based on a modified salting-out protocol. The Probe- qPCR assay using bcsp31 gene amplification showed an efficiency of 92.35%, with a slope of -3.52 reached in the standard curve. The qPCR assay detected 9.5% (n = 62/656; 95% CI: 7.3,12.0) of the animals with Brucella-DNA presence, and 62.5% (n = 25/40; 95% CI: 45.8,77.3) of the herds with Brucella-DNA presence. Using the SNP-based assay, all positive samples were identified as field Brucella strains. In the final regression model at the animal-level, five variables were associated with Brucella-DNA presence: the use of bulls for mating, recorded history of reproductive problems, pregnant cows, parlor milking, and cows belonging to farms ≤ 200 m from the main road. At the herd-level, two variables were associated with Brucella-DNA presence: recorded history of reproductive problems and bulls' use for mating. Given the fluctuant brucellosis prevalence in endemic areas, updated epidemiological studies are necessary to evaluate the disease dynamic, and if established prevention and control measures have been effective or need to be adjusted. The increase in the prevalence of brucellosis in animal reservoirs creates an important risk of transmission in humans.

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Section: Discussionmentioning

confidence: 67%

Section: Introductionmentioning

confidence: 99%

Epidemiology Aspects of Brucella-DNA Detection Using Real-time PCR from Bovine Whole Blood Samples in Colombia

Ramírez¹,

Santos²,

Paulino³

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Primer-Blast is a procedure for Primer design and comparison, which can effectively analyze the speci city of primers [18]. The combined use of these software can effectively improve the speci city and success rate of PCR, which is widely used [19]. The same process was used in this study and satisfactory results were obtained.…”

Section: Discussionmentioning

confidence: 97%

A New Specific Sequence to Distinguish B.canis From Other Brucella by PCR

Ye¹,

Yang²,

Li³

et al. 2021

Preprint

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Background: Brucellosis is a zoonotic disease worldwide. The increasing number of pet dogs has raised new risk of people getting canine brucella with absent or mild symptom. Besides, the canine brucellosis can be caused by other brucella species, so their infection could be omitted by the PCR method. The present PCR methods can only detect canine brucella, by which cases infected with other brucella would appear negative. It’s an urge to develop a specific PCR assay for detecting canine Brucellosis, Whether the pathogen was B.canis or any other Brucella.Resaults: a differential sequence of B.canis were found by genome comparison analysis and were analyzed by BLAST. Then a PCR method was established using specific primers in the sequence and tested for clinical application. It could detect canine brucellosis caused by B.canis or other Brucella species with 310-bp and 413-bp product, respectively. The developed PCR method had specificity for non-brucella and a sensitivity of 100 copies of Brucella DNA. The detection accuracy verified with spiked samples was 95.5% (21/22) for B.canis and 100% (22/22) for other brucella. Conclusions: The study found a specific sequence of B.canis and developed a PCR detection method to detect canine brucellosis caused by B.canis or other Brucella species. The method established in this study will more comprehensively detect the pathogen of canine brucellosis and provide important methods and means for preventing and controlling this disease.

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“…Primer-Blast is a procedure for primer design and comparison that can effectively analyze the specificity of primers (21). The combined use of this software can effectively improve the specificity and success rate of PCR assays, which are widely used (22). In this study, we went through that process and satisfactory results were obtained.…”

Section: Discussionmentioning

confidence: 98%

A specific reverse complement sequence for distinguishing Brucella canis from other Brucella species

Yang²,

Dong-Liang³

et al. 2022

Front. Vet. Sci.

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Canine brucellosis is primarily caused by Brucella canis, but other Brucella species can also cause the disease. Identifying sequences specific to B. canis and establishing PCR assays that can distinguish between B. canis and other Brucella species is essential to determine the etiology of canine brucellosis and the source of infection and to achieve effective control. We analyzed the gaps and SNPs of genomes I and II from B. canis strain RM6/66 and B. melitensis strain 16M using the Mauve genome alignment software, and the specificity of each of these differential regions was analyzed by BLAST. A 132 bp specific sequence was found between the DK60_915 (glycosyl hydrolase 108 family protein) and DK60_917 (aldose 1-epimerase) loci in B. canis chromosome 1. Further comparative analysis revealed that this is a reverse complement sequence between B. canis and other Brucella species. Then, three primers were designed based on the sequence that could detect B. canis with a 310 bp amplification product or other Brucella species with a 413 bp product. The PCR based on these primers had reasonable specificity and a sensitivity of 100 copies of Brucella DNA. The detection results for the blood samples of the aborted dogs showed a favorable accordance with the Bruce-ladder multiplex PCR assay. In conclusion, we found a specific reverse complement sequence between B. canis and other Brucella and developed a PCR method that allows a more comprehensive identification of the pathogen involved in canine brucellosis. These findings provide an effective means for preventing and controlling brucellosis.

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Combined nucleic acid assays for diagnosis of A19 vaccine‐caused human brucellosis

Cited by 13 publications

References 18 publications

Epidemiology Aspects of Brucella-DNA Detection Using Real-time PCR from Bovine Whole Blood Samples in Colombia

Epidemiology Aspects of Brucella-DNA Detection Using Real-time PCR from Bovine Whole Blood Samples in Colombia

A New Specific Sequence to Distinguish B.canis From Other Brucella by PCR

A specific reverse complement sequence for distinguishing Brucella canis from other Brucella species

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