Real-time PCR for identification of Brucella spp.: A comparative study of IS711, bcsp31 and per target genes

Bounaadja, Lotfi; Albert, David; Chénais, Benoı̂t; Hénault, Sylvie; Zygmunt, Michel S.; Poliak, Sylvie; Garin‐Bastuji, Bruno

doi:10.1016/j.vetmic.2008.12.023

Cited by 158 publications

(128 citation statements)

References 33 publications

Supporting

Mentioning

109

Contrasting

Unclassified

Order By: Relevance

“…It is noteworthy that different copy numbers of IS711 in various species and biovars affect the accuracy of the bacterial load estimation by real-time PCR assays that target this fragment, particularly in culture-negative cases. Bounaadja et al reported that the C T differences between IS711 and bcsp31 (⌬C T IS/bcsp) were 0.18 to 2.35 and 0.41 to 2.50 for the same B. abortus and B. melitensis DNA loads, respectively (31). Hence, the bcsp31 with one copy in brucellae can serve as an exact indicator for the detection of Brucella spp.…”

Section: Discussionmentioning

confidence: 99%

Efficient Diagnosis and Treatment Follow-Up of Human Brucellosis by a Novel Quantitative TaqMan Real-Time PCR Assay: a Human Clinical Survey

et al. 2014

View full text Add to dashboard Cite

cRapid and effective diagnosis of brucellosis is a challenge for clinicians. Even when diagnosis is on time and therapy is initiated, meticulous follow-up appointments are crucial for ensuring the efficacy of the treatment. Due to shortcomings of serological methods, molecular diagnosis, especially real-time PCR, is becoming a main approach in laboratory diagnostics. Thus, the development of efficient procedures and standardization of the PCR tests will have a great impact on the precise detection and quantification of bacterial DNA loads, which is valuable for the medical management of brucellosis patients. We developed a new TaqMan real-time PCR directed to bcsp31, a shared gene of the brucellae. The bcsp31 gene fragment was cloned into pJET1.2. Recombinant pJET1.2-bcsp31 was linearized by HindIII digestion, and the product was used for the preparation of a standard curve. A panel of Brucella spp. and non-Brucella pathogens was tested. No bacterial genomes other than those of the brucellae were detected. According to the results, specificity of the method was 100%. In a clinical assessment, the positive-control group comprised 37 patients with microbiologically confirmed brucellosis, and 25 healthy individuals served as the negative-control group. By the end of the treatment period, there was a significant decrease in the DNA load of the 37 brucellosis patients, which persisted for the 4 weeks of monitoring after treatment, suggesting that our proposed method is an efficient monitoring tool. Serum samples prior to any treatment were collected from the 25 serologically suspicious patients and assessed by our method; 72% of these patients tested positive for brucellosis.T he brucellae are facultative, intracellular, Gram-negative bacteria that infect animals and humans (1). Although it has been eradicated in some developed countries, brucellosis still remains a major problem worldwide. Diagnosis of the disease presents a major challenge to clinical and veterinary services (2, 3). Brucella spp. are able to survive within host cells, leading to relapses and focal complications (1). Early diagnosis of brucellosis results in early initiation of therapy, which plays a key role in the success of the control and eradication programs; nonetheless, accurate diagnosis requires reliable and sensitive diagnostic tools (4). The intracellular localization of the bacteria and the slow evolution of the disease hamper the usefulness of blood cultures (5). Despite being the gold standard, isolation of brucellae from blood culture or other sterile body fluids has a sensitivity of about 70% (4, 6). Serodiagnostic assays are principally based on antibodies against the Brucella lipopolysaccharide (LPS) and have high sensitivity but low specificity. The low specificity may be due to the crossreaction of antibodies with LPS from other bacterial species and subsensitive or high-immunity reactions, depending on the subclinical or endemic prevalence of the disease (4, 6, 7). The presence of IgG and IgM for months after therapy complicates...

show abstract

Section: Discussionmentioning

confidence: 99%

Efficient Diagnosis and Treatment Follow-Up of Human Brucellosis by a Novel Quantitative TaqMan Real-Time PCR Assay: a Human Clinical Survey

et al. 2014

View full text Add to dashboard Cite

show abstract

“…(Navarro et al, 2004). The BCSP31-PCR assay was carried out using standard procedure (Baily et al, 1992;Bounaadja et al, 2009), and IS711-PCR was done using the procedure described by Halling et al (1993). The cut off for the cycle threshold (Ct) values of real-time PCR positivity is 40.…”

Section: Real-time Pcrmentioning

confidence: 99%

Seroprevalence and Risk Factors for Brucellosis in a High-Risk Group of Individuals in Bangladesh

Rahman

Berkvens

Frétin

et al. 2012

Foodborne Pathogens and Disease

View full text Add to dashboard Cite

Brucellosis is an occupational hazard of livestock farmers, dairy workers, veterinarians, slaughterhouse workers, and laboratory personnel, all of whom are considered to belong to the high-risk occupational group (HROG). A study was undertaken to determine the seroprevalence of brucellosis, identify risk factors associated with brucellosis seropositivity, and detect Brucella at genus level using real-time polymerase chain reaction (PCR) among people in the HROG in the Dhaka division of Bangladesh. A sample of 500 individuals from the HROG was collected from three districts of Dhaka division of Bangladesh. A multiple random effects logistic regression model was used to identify potential risk factors. Two types of real-time PCR methods were applied to detect Brucella genus-specific DNA using serum from seropositive patients. The prevalence of brucellosis based on the three tests was observed to be 4.4% based on a parallel interpretation. The results of the multiple random effects logistic regression analysis with random intercept for district revealed that the odds of brucellosis seropositivity among individuals who had been in contact with livestock for more than 26 years was about 14 times higher as compared to those who had less than 5 years of contact with livestock. In addition, when the contact was with goats, the odds of brucellosis seropositivity were about 60 times higher as compared to when contact was with cattle only. Noticeable variation in brucellosis seropositivity among humans within the three districts was noted. All of the 13 individuals who tested positive for the serological tests were also positive in two types of real-time PCR using the same serum samples. Livestock farmers of brucellosis positive herds had a significantly higher probability to be seropositive for brucellosis. The study emphasized that contact with livestock, especially goats, is a significant risk factor for the transmission of brucellosis among individuals in the HROG.

show abstract

“…However, sensitivity of the omp2a LAMP assay was equivalent to TaqMan probe based real time PCR. The IS711 TaqMan probe based real time PCR has been reported as a highly sensitive method in detecting less than 10 fg of genomic DNA (three genome copies) of Brucella organisms [17,23,24].…”

Section: Resultsmentioning

confidence: 99%

Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay

et al. 2016

View full text Add to dashboard Cite

Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 9 10 1 CFU and 2.28 9 10 2 CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen.

show abstract

Real-time PCR for identification of Brucella spp.: A comparative study of IS711, bcsp31 and per target genes

Cited by 158 publications

References 33 publications

Efficient Diagnosis and Treatment Follow-Up of Human Brucellosis by a Novel Quantitative TaqMan Real-Time PCR Assay: a Human Clinical Survey

Efficient Diagnosis and Treatment Follow-Up of Human Brucellosis by a Novel Quantitative TaqMan Real-Time PCR Assay: a Human Clinical Survey

Seroprevalence and Risk Factors for Brucellosis in a High-Risk Group of Individuals in Bangladesh

Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay

Contact Info

Product

Resources

About