Search

Leptospirosis is an emerging zoonotic disease caused by bacterial species of the genus Leptospira. However, the regulatory mechanisms and pathways underlying the adaptation of pathogenic and non-pathogenic Leptospira spp. in different environmental conditions remain elusive. Leptospira biflexa is a non-pathogenic species of Leptospira that lives exclusively in a natural environment. It is an ideal model not only for exploring molecular mechanisms underlying the environmental survival of Leptospira species but also for identifying virulence factors unique to Leptospira's pathogenic species. In this study, we aim to establish the transcription start site (TSS) landscape and the small RNA (sRNA) profile of L. biflexa serovar Patoc grown to exponential and stationary phases via differential RNA-seq (dRNA-seq) and small RNA-seq (sRNA-seq) analyses, respectively. Our dRNA-seq analysis uncovered a total of 2726 TSSs, which are also used to identify other elements, e.g., promoter and untranslated regions (UTRs). Besides, our sRNA-seq analysis revealed a total of 603 sRNA candidates, comprising 16 promoter-associated sRNAs, 184 5ʹUTR-derived sRNAs, 230 true intergenic sRNAs, 136 5ʹUTR-antisense sRNAs, and 130 open reading frame (ORF)-antisense sRNAs. In summary, these findings reflect the transcriptional complexity of L. biflexa serovar Patoc under different growth conditions and help to facilitate our understanding of regulatory networks in L. biflexa. To the best of our knowledge, this is the first study reporting the TSS landscape of L. biflexa. The TSS and sRNA landscapes of L. biflexa can also be compared with its pathogenic counterparts, e.g., L. borgpetersenii and L. interrogans, to identify features contributing to their environmental survival and virulence. [ABSTRACT FROM AUTHOR]

Asymmetric PCR is one of the most utilized strategies in ssDNA generation towards DNA aptamer generation due to its low cost, robustness and the low amount of starting template. Despite its advantages, careful optimization of the asymmetric PCR is still warranted to optimize the yield of ssDNA. In this present study, we have developed an extensive optimization pipeline that involves the optimization of symmetric PCR initially followed by the optimization of asymmetric PCR. In the asymmetric PCR, optimization of primer amounts/ratios, PCR cycles, annealing temperatures, template concentrations, Mg2+/dNTP concentrations and the amounts of Taq Polymerase was carried out. To further boost the generation of ssDNA, we have also integrated an additional single-stranded DNA generation method, either via lambda exonuclease or biotin-streptavidin-based separation into the optimization pipeline to further improve the yield of ssDNA generation. We have acquired 700 ± 11.3 and 820 ± 19.2 nM for A-PCR-lambda exonuclease and A-PCR-biotin-streptavidin-based separation, respectively. We urge to develop a separate optimization pipeline of asymmetric PCR for each different randomized ssDNA library before embarking on any SELEX studies. [ABSTRACT FROM AUTHOR]

Although the multi-copy and specific element IS 6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS 6110-negative strains suggested that false negative may occur if IS 6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS 6110 and an IS-like elements designated as B 9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS 6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary. [ABSTRACT FROM AUTHOR]

Purification of RNA fragments from a complex mixture is a very common technique, and requires consideration of the time, cost, purity and yield of the purified RNA fragments. This study describes the fastest method of purifying small RNA with the lowest cost possible, without compromizing the yield and purity. The technique describes the purification of small RNA from polyacrylamide gel, resulting in a good yield of small RNA with minimum experimental steps in avoiding degradation of the RNA, obviating the use of ethidium bromide and phenol-chloroform extraction, as well as siliconized glass wools to remove the polyacrylamide gel particles. The purified small RNA is suitable for a wide variety of applications such as ligation, end labelling with radio isotope, RT-PCR (Reverse Transcriptase-PCR), Northern blotting, experimental RNomics study and also Systematic Evolution of Ligands by Exponential Enrichment (SELEX). [ABSTRACT FROM AUTHOR]