A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp

Abstract

Background: Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT.

Insights into Leptospirosis, a Neglected Disease

“…One of them is the 16s rDNA from which a 631 bp product [Hookey, 1992] or a 330 bp product (Senthilkumar et al 2001) can be amplified. A real-time PCR method based on 16S rDNA was developed that could be used on samples without the need for prior isolation and culture [Smythe et al, 2002]. Another popularly used method [Gravekamp et al, 1993] [Taylor et al, 1997] that consists of the immuno-magnetic separation of leptospires from inhibitors in frozen formalin-fixed bovine urine prior to PCR detection that resulted in a marked improvement in the detection of leptospires in urine samples.…”

Hamster Model of Leptospirosis

“…For some types of research, such as antibiotic prophylaxis and treatment studies, a quantitative measure of leptospiral tissue burden is desirable. Amplification of a 87-bp region of the leptospiral 16S gene by quantitative (TaqMan) PCR has been shown to have a mean detection limit of two cells (Smythe et al, 2002).…”

Detection of pathogenic leptospires by real-time quantitative PCR

“…Thus it was important to demonstrate the stability of leptospiral DNA added to a range of blood collection tubes and to urine. Vacutainer tubes containing citrate or EDTA gave optimal results up to 5 days after addition of blood containing viable leptospires; tubes containing heparin were inhibitory, as reported previously (Smythe et al, 2002), and also those containing sodium polyanetholesulfonate or saponin. Leptospiral DNA was detectable in human urine samples stored at ambient temperature for prolonged periods in DNA/RNA Protect containers.…”

“…The LipL32 primers can also be used in a conventional PCR assay (data not shown). The analytical sensitivity of the assay was 3 genome copies per reaction in blood and approximately 10 genome equivalents per reaction in urine, comparable to a real-time assay which uses a 16S rRNA gene target (Smythe et al, 2002).…”

“…Despite their wide use these assays have limitations: that described by Merien et al (1995) is a genus-specific assay which amplifies both pathogenic and non-pathogenic serovars, while the approach described by Gravekamp et al (1993) and evaluated by Brown et al (1995) requires amplification of two distinct targets in order to detect all species containing pathogens. More recently, a realtime PCR was developed using TaqMan chemistry (Smythe et al, 2002) which targeted an 87 bp section of the 16S rRNA gene of Leptospira spp. In this study, we report the development of a real-time assay using SYBR green chemistry, in which the target is the gene for the major outer-membrane lipoprotein LipL32 (Haake et al, 2000), which appears to be an important virulence factor (Yang et al, 2002), confined to pathogenic strains of all Leptospira spp.…”