Construction of DNA ladder based on 16S rRNA gene of Bacillus subtilis using touchdown PCR technique

Aim@#DNA molecular size markers or DNA ladders play a vital role in molecular biology laboratories where DNA electrophoresis experiments are usually conducted. This study aimed to produce a 100 bp DNA ladder at laboratory scale, which could be applied to determine the size of DNA fragments in molecular biology experiments.@*Methodology and results@#In this study, 14 primers including 4 forwards and 10 reverses were designed based on the 16S rRNA gene sequence of Bacillus subtilis. These primers were able to amplify 10 DNA fragments with accurate sizes from 100 to 1000 bp. Furthermore, touchdown PCR was involved to maximize the specificity and yield of PCR products. Ten DNA fragments with the sizes including 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 bp were synthesized, and such bands were equivalent with commercial DNA ladders. Moreover, the quantity and quality of PCR products were measured using a nanodrop spectrophotometer. The optimal concentration ratios between such fragments (100- 1000 bp) were 800, 300, 150, 150, 500, 50, 50, 50, 50 and 50 (ng/µL), respectively. These ratios showed the clear and high resolution on 1.5% agarose gel. @*Conclusion, significance and impact of the study@#The results indicated that 16S rRNA gene of B. subtilis was a potential material for DNA ladder preparation due to the multiple copies number of this gene. Furthermore, in combination with touchdown PCR, the nonspecific bands were reduced, and the products could be used directly without the need of purification step.

Adaptive experiment of VNUKOVO-32 vcell culture rabies vaccine strain in cell lines

Vnukovo-32 (cell culture rabies virus vaccine strain) was propagated experimentally in primary hamster kidney cell (PHKC), primary Chick embryo fibroblast cell (CEF) and human diploid cell (WI-38). These 3 cell lines were infected with Vnukovo-32 by the same mount of infection (MOl) - lLD50/l000 cells. The result showed that the Vnukovo-32 was adapted on all experimental cell lines, met the criteria of master seed (the titer ranging 6.4-7.5 log LD50/ml), and reached the highest titer on the 7th - 9th day postinfection in three cell groups. Moreover, the highest titer of Vnukovo-32 was met in human diploid cell (WI-38), then in PHCK, and the lowest was in CEF. From these results, it is suitable to use WI-38 to propagate Vnukovo-32 for master and working seed. The procedure for propagation of Vnukovo-32 is set up.

Quick diagnosis of japanese encephalitis by MAC-ELISA technique

On 138 serum samples taken in the first 7 days of acute syndrome of brain, by MAC-ELISA technique 95 samples were determined as positive, 68.84% was positive. 43 samples taken in the first 7 days of the disease there was no detected antivirus IgM antibody but this could not concluded as negative, but may be a late antibody response or may be another cause of acute brain syndrome. For assuring the accuracy of the diagnosis, the samples taken in the first 7 days of the disease must be taken again after 7 day to verify the results.

Evaluation of bioavailability of ketoprofen 200 mg long acting tablets

In vitro bioavailability of ketoprofen 200mg long acting tablets by the oral were studied. Using basket dissolution tester at 120rpm. Released ketoprofen was determined by a UV – spectrophotometer at 260nm. Ketoprofen plasma levels on carefully chosen 6 healthy volunteers were within treatment which reached from 4 to 36 hours. 200mg ketoprofen LP tablets gradually released ketoprofen within 24 hours and released about 80% after 18 hours. Pharmacological parameter of ketoprofen 200mg long acting tablets have the same value compared with the results which have been published