INTRODUCTION:: This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains.
METHODS:: Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively.
RESULTS:: The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water.
CONCLUSIONS:: Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.
Leptospira species were studied in water and soils from selected urban sites in Malaysia. A total of 151 water (n=121) and soil (n=30) samples were collected from 12 recreational lakes and wet markets. All samples were filtered and inoculated into semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media supplemented with additional 5-fluorouracil. The cultures were then incubated at 30°C and observed under a dark field microscope with intervals of 10 days. A PCR assay targeting the rrs gene was used to confirm the genus Leptospira among the isolates. Subsequently, the pathogenic status of the isolates was determined using primer sets G1/G2 and Sapro1/Sapro2, which target the secY and rrs genes, respectively. The isolates were identified at serogroup level using the microscopic agglutination test (MAT) while their genetic diversity was assessed by pulsed field gel electrophoresis (PFGE). Based on dark field microscopy, 23.1% (28/121) water and 23.3% (7/30) soil cultures were positive for Leptospira spp. Of the 35 positive cultures, only 8 were pure and confirmed as Leptospira genus by PCR assay. Two out of 8 isolates were confirmed as pathogenic, 5 were saprophytic and one was intermediate. These 8 isolates were negative for the 25 reference hyperimmune rabbit sera tested in the MAT. PFGE showed that all 8 of these environmental Leptospira spp. were genetically diverse. In conclusion, the presence of pathogenic Leptospira spp. in the urban Malaysian environment may indicate and highlight the importance of water screening, especially in recreational lakes, in order to minimize any chance of Leptospira infection.
This paper presents the development of tapered optical fiber sensor to detect a specific Leptospira bacteria DNA. The bacteria causes Leptospirosis, a deadly disease but with common early flu-like symptoms. Optical single mode fiber (SMF) of 125 μm diameter is tapered to produce 12 μm waist diameter and 15 cm length. The novel DNA-based optical fiber sensor is functionalized by incubating the tapered region with sodium hydroxide (NaOH), (3-Aminopropyl) triethoxysilane and glutaraldehyde. Probe DNA is immobilized onto the tapered region and subsequently hybridized by its complementary DNA (cDNA). The transmission spectra of the DNA-based optical fiber sensor are measured in the 1500 to 1600 nm wavelength range. It is discovered that the shift of the wavelength in the SMF sensor is linearly proportional with the increase in the cDNA concentrations from 0.1 to 1.0 nM. The sensitivity of the sensor toward DNA is measured to be 1.2862 nm/nM and able to detect as low as 0.1 fM. The sensor indicates high specificity when only minimal shift is detected for non-cDNA testing. The developed sensor is able to distinguish between actual DNA of Leptospira serovars (Canicola and Copenhageni) against Clostridium difficile (control sample) at very low (femtomolar) target concentrations.
Melioidosis and leptospirosis, caused by two different bacteria, Burkholderia pseudomallei and Leptospira spp., are potentially fatal infections that share a very similar spectrum of clinical features and cause significant mortality and morbidity in humans and livestock. Early detection is important for better clinical consequences. To our knowledge, there is no diagnostic tool available to simultaneously detect and differentiate melioidosis and leptospirosis in humans and animals. In this study, we described a duplex TaqMan probe-based qPCR for the detection of B. pseudomallei and Leptospira spp. DNA. The performance of the assay was evaluated on 20 B. pseudomallei isolates, 23 Leptospira strains, and 39 other microorganisms, as well as two sets of serially diluted reference strains. The duplex qPCR assay was able to detect 0.02 pg (~ 4 copies) Leptospira spp. DNA and 0.2 pg (~ 25.6 copies) B. pseudomallei DNA. No undesired amplification was observed in other microorganisms. In conclusion, the duplex qPCR assay was sensitive and specific for the detection of B. pseudomallei & Leptospira spp. DNA and is suitable for further analytical and clinical evaluation.