ABSTRACT
Background: The quantitative assays are important tests in the management of patients with HIV-1/AIDS. The important step in developing the assay is the availability of the standard HIV-1 RNA. For this purpose, we optimized in vitro HIV-1 RNA transcription to produce the standard HIV-1 RNA. Methods: The HIV-1 DNA was amplified from pNL43 by PCR using a primer pair that was specific for conserved region of HIV-1 Gag gene. The PCR product was further cloned into pBluescript II KS. The recombinant plasmid was restricted with EcoRI enzyme. Then, the linearized plasmid was used as template for RNA transcription. RT-PCR and PCR were performed simultaneously for confirmation of synthesized RNA fragment. Results: A 115 bp DNA of HIV-1 Gag gene has been cloned into pBluescript II SK with the exact true orientation. The reaction of the RNA transcription was also successfully performed. The RNA transcripts have been confirmed and showed the accuracy of the transcripts. Conclusion: We successfuly constructed the recombinant plasmid containing a conserved region of HIV-1 Gag gene, and the HIV-1 RNA has been transcribed in vitro as well.
Subject(s)
HIV Infections , RNA Processing, Post-TranscriptionalABSTRACT
Aim: Since culture method is time-consuming and has low sensitivity, we developed a duplex PCR (dPCR) assay for the detection of Legionella sp. and L. pneumophila in cooling tower samples. We used culture method as a gold standard. Methods: Optimization of dPCR method was performed to obtain an assay with high sensitivity and specifi city. The optimized method was used to detect Legionella sp. dan L. pneumophila in 9 samples obtained from 9 buildings in Jakarta. For culture method, the bacteria were grown or isolated on selective growth factor supplemented-buffered charcoal yeast extract (BCYE) media. Results: Of 9 samples tested by dPCR assay, 6 were positive for Legionella species,1 was positive for L. pneumophila, and 2 showed negative results. For the same samples, no Legionella sp. was detected by the culture method. Conclusion: dPCR assay was much more sensitive than the culture method and was potentially used as a rapid, specifi c and sensitive test for routine detection of Legionella sp. dan for L. pneumophila in water samples.
Subject(s)
Legionella pneumophila , Legionnaires' DiseaseABSTRACT
Aim A spesific and rapid diagnosis such as RT-PCR asay is the most needed to minimize transmission of HIV-1 infection. Therefore, in this study we developed the RT-PCR assay that was spesifi c against the gag gene of HIV-1. Methods The developed RT-PCR assay was evaluated against 46 specimens that were obtained from voluntary counseling and testing for HIV (VCT) in Rumah Sakit Umum Pemerintah (RSUP) Sanglah, Bali. To get the sensitivity and specifi city of RT-PCR assay, the results of assays were compared with the results of commercially serologic tests that were commonly used in Indonesia. Results The RT-PCR assay could detect 21 of 26 serologic test-positive specimens and showed 19 negative results of 20 serologic test-negative specimens. There was one specimen that was positive in RT-PCR but negative in serologic assay, which might depict a true yield at particular condition when the serologic assay was unable to detect. Five serologic positive-test specimens were negative by RT-PCR that was possibly caused by low detection level of the assay. Conclusion The RT-PCR assay is potential to be used for the detection of HIV-1 infection with a sensitivity and specificity of 80.8% and 95.0% respectively.
Subject(s)
HIV InfectionsABSTRACT
Sequence analysis was conducted on structural and non-structural genes of 7 strains of dengue virus type-3 (DENV-3 virus) isolated in Indonesia and Thailand in the year 1973, 1994, and 1998 from patients with different clinical manifestations. In general, sequence similarity among isolates was greater than 93%, indicating that the mutation rate of DENV-3 circulating in this region was not more than 7% in the last 3 decades and suggesting that sequences that may responsible for viral architectures and/or biological function were strictly conserved. Mutations unique to viral strains associated with specific clinical manifestations were not found. Alignment of PrM/M and E nucleic acid sequences followed by parsimony analysis of sequences obtained in this study and published elsewhere allowed generation of phylogenetic trees, demonstrating that DENV-3 strains isolated in Indonesia in 1998 belonged to a separate cluster (subtype 2) from those isolated between 1973-1985 (subtype 1).