2511st International Conference on Molecular Diagnostic and Biomarker Discovery/Asian Pac J Trop Dis 2014; 4(3): 223-252

Construction of vectors for tight regulation and repression of protein expressiondoi: 10.1016/S2222-1808(14)60566-1 襃 2014 by the Asian Pacific Journal of Tropical Disease. All rights reserved.

Chee Fah Wong1, Raja Noor Zaliha Raja Abd. Rahman2, Mahiran Basri2 and Abu Bakar Salleh2

1Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjung Malim, Perak, Malaysia2Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

ABSTRACTIntroduction: Protein expression by means of vector construction may facilitate in the synthesis of protein in sufficient amount for characterization, quorum sensing and molecular diagnostic studies. However, significant amount of protein also can be expressed even under non-inducing and repressed conditions when using many of the vectors available nowadays. In this communication, we report on the design of several genetic elements which resulted in more regulable and thus more tightly repressible protein expression system when grown under repressed conditions.Objective: To construct vectors for tight regulation and repression of protein expression.Methods: The genetic tools were generated via PCR and inserted into the newly constructed vectors by restriction endonucleases. Analysis of the constructed vectors was carried out by restriction endonuclease digestion and DNA sequencing. Elastase, on the other hand, was used as a model protein to test on the efficiency of the constructs. Thus, the resulting protein expression level was determined using elastinolytic assay (Ohman et al., 1980).Results & Discussion: The constructs harboring a PT7-dual operator system have greatly regulated and reduced the level of basal protein expression. In addition, the presence of a repressor gene has further assisted in controlling the expression level upon induction in several heterogeneous host systems.Conclusion: The ‘tightness’ and repression effects of the genetic elements in these constructs have lent remarkable support in expression of foreign genes in bacterial host systems.

In-house Loop Amplification (LAMP) method for the diagnosis of Salmonella Paratyphi A in low-resource settingsdoi: 10.1016/S2222-1808(14)60567-3 襃 2014 by the Asian Pacific Journal of Tropical Disease. All rights reserved.

Maizan M. 1, Nur Eliana S. 1, Zawiyah D. 1, Julia A. 1, Faizul-Rahman S. 1, Zaidah AR2 , Azura H. 3, Aziah I1 and Asma I

1

1Institute for Research in Molecular Medicine (INFORMM)2School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kota Bharu, Kelantan, Malaysia3Hospital Raja Perempuan Zainab II, 15586 Kota Bharu, Kelantan, Malaysia.

ABSTRACTIntroduction: Salmonella Paratyphi A (S. Paratyphi A) causes paratyphoid fever, a disease endemic in developing countries. It is transmitted via the faecal-oral route, mainly through contaminated food and water. The prevalence or incidence of the disease is increasing especially in China, India and Asia. Current diagnosis of S. Paratyphi A is via culture and molecular methods such as PCR. However, these methods do not offer a rapid, simple, and cost-effective detection of S. Paratyphi A especially for use in resource-limited settings.Objective: The study was aimed at developing an in-house LAMP method as a rapid, sensitive, specific and cost-effective detection of S. Paratyphi A.Methods: An In-house LAMP method was developed and optimized for the detection of S. Paratyphi A using primers that were designed based on intergenic region of SSPA1723a and SSPA1723 gene of S. Paratyphi A. The primers’ specificity was tested on 60 bacteria strains consisting of 25 S. Paratyphi A, 20 other Salmonella serovars, and 15 non-Salmonella bacteria which were obtained from Institute for Research in Molecular Medicine (INFORMM) culture bank. Detection limit of LAMP was determined using 10-fold serial dilutions of S. Paratyphi A strain DNA and compared with culture and PCR results. The assay was further evaluated on 60 BACTEC blood culture broths suspected of S. Paratyphi A. The results were compared with those obtained by PCR assay and culture method.Results & Discussion: In-house LAMP method was successfully established and optimized. The sensitivity of the assay was determined at 200 CFU. This LAMP assay gave positive result to all the 25 S. Paratyphi A isolates and negative to 20 other Salmonella and 15 non-Salmonella bacteria. This assay detected 8 samples as positive and 52 samples as negative for S. Paratyphi A. These results were similar to those results obtained by PCR assay and culture methods.Conclusion: An in-house LAMP method was established in this study and could potentially be used as a rapid, sensitive, specific and cost-effective detection method for S. Paratyphi A especially in low-resource settings. However this LAMP method is only recommended for screening purposes and need to be further confirmed with gold standard method which is culture method and PCR.