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

Abstract

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.

Keywords: Brucella; TaqMan real-time-polymerase chain reaction; bacteriological results; conventional polymerase chain reaction; diagnostic sensitivity; diagnostic specificity.

Figure-1

Diagnostic sensitivity, specificity, performance indices, accuracy, and area under the curve of real-time polymerase chain reaction (PCR) versus conventional PCR targeting Brucella DNA in cattle sera.

Figure-2

Standard curve of TaqMan real-time polymerase chain reaction targeting Brucella bscp31 gene plotting cycle threshold values versus log template concentrations.

Figure-3

Amplification curves of TaqMan real-time polymerase chain reaction for the detection of the DNA of the Brucella genus in cattle sera.

Figure-4

Detection of IS711 gene specific for genus Brucella by conventional polymerase chain reaction (PCR). Lane 1, PCR marker; Lane 2(P), B. melitensis reference strain Ether; lane3 (N), control negative; Lane 4 (sample 61), negative sample; lane 5-13 (samples No. 62, 63, 64, 65, 66, 67, 68, 69, 70), Brucella positive samples.

Figure-5

Receiver operating characteristic curves reflecting the diagnostic accuracy of TaqMan real-time polymerase chain reaction (PCR) versus the conventional PCR used to detect Brucella DNA in cattle sera.