A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood, and feces from marine mammals

Abstract

Abstract. Brucellosis has emerged as a disease of concern in marine mammals in the last 2 decades. Molecular detection techniques have the potential to address limitations of other methods for detecting infection with Brucella in these species. Presented herein is a real-time polymerase chain reaction (PCR) method targeting the Brucella genus-specific bcsp31 gene. The method also includes a target to a conserved region of the eukaryotic mitochondrial 16S ribosomal RNA gene to assess suitability of extracted DNA and a plasmid-based internal control to detect failure of PCR due to inhibition. This method was optimized and validated to detect Brucella spp. in multiple sample matrices, including fresh or frozen tissue, blood, and feces. The analytical limit of detection was low, with 95% amplification at 24 fg, or an estimated 7 bacterial genomic copies. When Brucella spp. were experimentally added to tissue or fecal homogenates, the assay detected an estimated 1-5 bacteria/µl. An experiment simulating tissue autolysis showed relative persistence of bacterial DNA compared to host mitochondrial DNA. When used to screen 1,658 field-collected marine mammal tissues in comparison to microbial culture, diagnostic sensitivity and specificity were 70.4% and 98.3%, respectively. In addition to amplification in fresh and frozen tissues, Brucella spp. were detected in feces and formalin-fixed, paraffin-embedded tissues from culture-positive animals. Results indicate the utility of this real-time PCR for the detection of Brucella spp. in marine species, which may have applications in surveillance or epidemiologic investigations.

Optimization and Validation of a Diagnostic Real-Time PCR for Bovine Brucellosis

“…Also numerous reports mentioned the use of bcsp31 for specific identification of genus Brucella from seropositive, active, relapsing, chronic cases in humans (Kattar et al, 2007;Mitka et al, 2007;Queipo-Ortuno et al, 2008). Recently the same gene target has been used specifically to detect Brucella in human serum, blood and cerebro-spinal fluid (Debeaumont et al, 2005;Colmenero et al, 2011;Sohrabi et al, 2011), in buffalo milk (Amoroso et al, 2011) and in clinical tissues from seals (Sidor et al, 2013).…”

“…Most of these reports indicated above were based on clinical studies derived from samples from human cases, and the selected gene targets were bcsp 31 or the IS711 element. But fewer number of such studies have been conducted on animals or animal products (Amoroso et al, 2011;Sohrabi et al, 2011;Dehkordi et al, 2012;Sidor et al, 2013;El Behiry et al, 2014;Dean et al, 2014). However, reports on validation of qPCR for brucellosis are still scarce in the literature (Probert et al, 2004;Debeaumont et al, 2005;Amoroso et al, 2011;Sidor et al, 2013).…”

“…However, reports on validation of qPCR for brucellosis are still scarce in the literature (Probert et al, 2004;Debeaumont et al, 2005;Amoroso et al, 2011;Sidor et al, 2013). Most of these reports are based on screening of DNA panels covering an array of Brucella and non-Brucella strains (Bogdanovich et al, 2004;Probert et al, 2004), but only a few on actual clinical samples from animal or human origin (Amoroso et al, 2011;Sidor et al, 2013). In recent times there are only two reports of a validated Brucella genus specific qPCR; one from marine mammals (Sidor et al, 2013) and another from human serum (Debeaumont et al, 2005).…”

“…But fewer number of such studies have been conducted on animals or animal products (Amoroso et al, 2011;Sohrabi et al, 2011;Dehkordi et al, 2012;Sidor et al, 2013;El Behiry et al, 2014;Dean et al, 2014). However, reports on validation of qPCR for brucellosis are still scarce in the literature (Probert et al, 2004;Debeaumont et al, 2005;Amoroso et al, 2011;Sidor et al, 2013). Most of these reports are based on screening of DNA panels covering an array of Brucella and non-Brucella strains (Bogdanovich et al, 2004;Probert et al, 2004), but only a few on actual clinical samples from animal or human origin (Amoroso et al, 2011;Sidor et al, 2013).…”

“…Most of these reports are based on screening of DNA panels covering an array of Brucella and non-Brucella strains (Bogdanovich et al, 2004;Probert et al, 2004), but only a few on actual clinical samples from animal or human origin (Amoroso et al, 2011;Sidor et al, 2013). In recent times there are only two reports of a validated Brucella genus specific qPCR; one from marine mammals (Sidor et al, 2013) and another from human serum (Debeaumont et al, 2005). In present study we describe the validation of the qPCR assay for identification of genus Brucella from clinical samples of bovine and bubaline origin from India, as prescribed in the guidelines of the validation and quality control of polymerase chain reaction methods used for diagnosis of infectious diseases (OIE, 2008) and according to requirements of the minimum information necessary for evaluating qPCR experiments (Bustin et al, 2009).…”