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Background: Traditionally, the most common diagnostic approach used for diagnosing leptospirosis was the demonstration of immune-seroconversion in acute and convalescent patient serum samples. Recently, a variety of molecular techniques, including conventional and real-time polymerase chain reaction (PCR), have been developed for the specific detection of pathogenic bacteria from the genus Leptospira. PCR is a sensitive, specific, and rapid technique that has been successfully used to detect several microorganisms; including those of clinical significance. Methods: In this study, we developed a multiplex PCR (mPCR) assay for detecting Leptospira's DNA. The mPCR assay detects both the 16S rRNA gene and the major outer membrane lipoprotein gene, which is known as LipL32. Representative serovars were tested from 10 species of Leptospira and 23 other species of bacteria. Results: A positive result was obtained from all leptospiral serovars. The amplification sensitivity for the multiplex assay was 21.8 pg and 1 x 103 leptospires/ml. This mPCR assay has the potential to facilitate a rapid and sensitive diagnosis for acute leptospirosis. Conclusion: The mPCR assay developed in this study can be used for the early detection of leptospirosis. The LipL32 gene could also serve as another target to aid in the efficient detection of leptospiral infection because using 2 sets of primers in mPCR increases the sensitivity and specificity of the test. [ABSTRACT FROM AUTHOR]

Background: A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. Methods: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. Results: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. Conclusion: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication. [ABSTRACT FROM AUTHOR]