RESUMEN
Many techniques have been developed for extracting DNA, but most are often complex, time-consuming, and/or expensive. In this study, we describe a simple, rapid and cost-effective technique for preparing DNA template for PCR. This technique involves 0.1 M potassium hydroxide treatment at 100°C for 10 min followed by centrifugation. The suspended centrifuged sediments were shown as excellent templates for PCR. Templates prepared using this technique worked for diverse microorganisms belonging to bacteria, fungi and oomycetes and their amplification efficiencies were comparable to/better than those prepared using common but relatively more complex, time-consuming, and/or expensive methods, including commercial DNA extraction kits. Furthermore, this technology is suitable for high-throughput batch processing and for amplifications of long DNA fragments. Flow cytometry and scanning electronic microscopy analyzes showed that this technique generated primarily damaged cells and cell-bound DNA, not free naked DNA. This technique provides a great convenience for simple PCR template preparation.
RESUMEN
The surveillance of multidrug-resistant (MDR) H58 typhoid is highly important, especially in endemic areas. MDR strain detection is needed by using a simple PCR technique that only uses a pair of primers. This is conducted considering the detection of Salmonella Typhi strains that have been carried out so far are only using antimicrobial sensitivity tests to determine microbial resistance phenotypically and to determine genotypically using complex molecular techniques. We aimed to analyse the existence of Salmonella Typhi MDR H58 in patients with typhoid fever in Makassar, Indonesia. A total of 367 blood samples of typhoid fever patients were collected from April 2018 until April 2019. The blood sample was cultured, then confirmed via simple PCR. All of the confirmed samples were tested for susceptibility against antibiotics and molecularly analysed for MDR H58 existence using a simple PCR technique. We found 7% (27/367) of the samples to be positive by both blood culture and PCR. All 27 isolates were found to be sensitive to sulfamethoxazole/trimethoprim. The lowest drug sensitivities were to amoxicillin, at one (3.7%) of 27 isolates, and ampicillin, at 13 (48.1%) of 27 isolates. Salmonella Typhi H58 PCR results showed that one (3.7%) of 27 isolates carried a positive fragment of 993 bp that led to the H58 strain, since the deletion flanks this fragment. The isolate was also found to be resistant to amoxicillin and fluoroquinolone according to a sensitivity test. Further molecular analysis needs to be conducted to examine the single isolate that carried the 933 bp fragment.
RESUMEN
Bovine Tropical Theileriosis (BTT) is an important vector-borne protozoan disease that imposing serious constraints on the health and productivity of domestic cattle. It is matter of common fact that following recovery from primary infection, cattle become persistent carriers and act as reservoirs of infection thereby, playing a critical role in disease epidemiology. The present study describes the comparative diagnostic efficiency of simplex PCR, duplex PCR and RAPD assays for detection of Theileria annulata in cattle. An optimized simple PCR and duplex PCR assay were established using TAMS F/R as primer sets encoding for 721 bp amplicon alongside a RAPD with arbitrary primer coding for 963 bp product of T. annulata. The simple PCR and duplex PCR detected pathogen with almost same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen without nonspecific amplifications. RAPD failed to give comparable results and suffered from limitations of sensitivity as well as specificity. The developed assays may be seen as a good tool for epidemiological studies aiming at assessing the burden of chronic infections and improving control of the associated diseases in endemic regions.