Evaluation of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle

Abstract

A study was performed to evaluate the previously described PCR (C. Romero, C. Gamazo, M. Pardo, and I. López-Goñi, J. Clin. Microbiol. 33:615-617, 1995) for the diagnosis of brucellosis in dairy cattle. Milk samples from 56 Brucella milk culture-positive cattle and from 37 cattle from Brucella-free herds were examined for Brucella DNA by PCR and for specific antibodies by an indirect enzyme-linked immunosorbent assay (ELISA). The specificities of both tests were 100% when testing the milk samples from Brucella-free cattle. The milk samples from 49 infected cattle were positive by PCR (87.5% sensitivity), and 55 were positive by ELISA (98.2% sensitivity). A PCR-positive sample was negative by ELISA, and 7 ELISA-positive samples were PCR negative, yielding an observed proportion of agreement of 0.91 for the two tests. Although the results suggest that ELISA is a better screening test than PCR, the combined sensitivity of the two assays was 100%, and their simultaneous application could be more useful than one test alone for a rapid screening of brucellosis in dairy cattle.

Comparison of three different PCR methods for detection of Brucella spp. in human blood samples

“…All these assays have used Brucella DNA of pure cultures [5^9]. But only a few of these primers have been used in animal [8,10,11] and human samples [8,12,13], and no studies have been carried out to compare sensitivity between them. The diagnosis of human brucellosis from blood is routinely performed in the presence of human genomic DNA and, to date, nobody has studied whether the sensitivity of one primer pair is the same in the absence or presence of human DNA.…”

Outbreak of Brucellosis in Domestic Elk in Korea

“…All nine of the isolates we obtained generated 905‐bp amplicons upon 16S rRNA‐based Brucella genus‐specific PCR, which indicates that they all belonged to the Brucella genus. This Bru16‐PCR assay is Brucella genus‐specific, although it does show cross‐reactivity with Ochrobactrum anthropi biotype D. However, this bacterium is usually found as an opportunistic infection in immuno‐compromised hosts, which is a situation that is not likely to be confused with brucellosis (Bricker and Halling, 1994; Romero et al., 1995a,b). All nine isolates also generated 498‐ and 178‐bp amplicons in the differential Brucella species AMOS‐PCR assay, which amplifies multi‐copy element IS 711 and has been improved by the addition of the eri gene as an amplification target (Bricker, 2002; Bricker and Halling, 1994, 1995; Ewalt and Bricker, 2000).…”

“…The genomic DNA of the isolates was extracted by using a DNeasy blood & tissue kit (Qiagen Korea Ltd., Seoul, Korea) according to the manufacturer’s instructions and stored at 4°C until further use. 16S rRNA‐based Brucella genus‐specific PCR (Bru16‐PCR) and IS 711 and eri gene‐based differential Brucella species AMOS‐PCR were performed for molecular verification of the Brucella isolates (Bricker and Halling, 1994; Ewalt and Bricker, 2000; Ramesh et al, 1999; Romero et al., 1995a,b). The sequences of the primers that were used and the size of the resulting amplicons are given in Table 1.…”

“…Polymerase chain reaction amplification was performed by using an AccuPower PCR premix (Bioneer Co, Deajeon, Korea). The PCR conditions were as described previously (Ewalt and Bricker, 2000; Romero et al., 1995a,b). Amplification was performed by using a thermocycler (Biometra, Göttingen, Germany).…”

Molecular detection of Brucella spp. from broth culture of clinical samples in Nigeria: Its role in vaccine quality control

“…DNA was extracted from broth cultures of Brucella using a modified method of extraction of Brucella DNA according to Romero et al (1995). 500 ml of the broth culture was aliquoted into a micro centrifuge tube; 500 µl of buffer 1 (20 mM NaCl, 20 mM EDTA, 20 mMTris-HCl pH = 7.5, 0.5% Triton x -100) was added and left on ice for 30 min.…”