Applicability of an oligonucleotide probe in radioisotope 32P-based dot blot hybridization for detection of hepatitis C virus in large sample numbers: a preliminary study.

Background: This study aimed to design and analyze the applicability of an oligonucleotide probe in radioisotope 32P-based dot blot hybridization for detection of hepatitis C virus. Methods: Forty-six of plasma samples were used. The plasma was extracted to obtain viral RNA genome as template for RT-PCR and the amplicon was used for nested PCR. Twenty-four HCV genomes were retrieved from GeneBank DNA sequence and alignment was performed by Bio Edit Software version 7.0.9.0. An oligonucleotide probe was designed based on a highly conserved region that is located on internal sequence between two primers used for nested PCR. Blast analysis on GeneBank was performed to obtain homology of the oligonucleotide for HCV. The oligonucleotide was then labeled with 32P and dot blot hybridization was applied for nested PCR products. DNA Sequencing was performed to confirm the amplicon and dot blot hybridization results. Results: Blast analysis showed high homology (100%) for HCV. Nested PCR resulted in three patterns of DNA fragments representing HCV genotypes 1, 2, and 3, respectively. The primers used in nested PCR were not specific and resulted in DNA fragments difficult to be interpreted. Dot blot hybridization using the designed oligonucleotide showed high intensity dots. All nested PCR fragments showed the dot blot positive. The dot blot results were in accordance with DNA sequencing that confirmed three patterns of DNA fragments as different HCV genotypes. Conclusion: The oligonucleotide showed excellent bioinformatically criteria. 32P-based dot blot hybridization yielded high intensity dots and was easier to be interpreted than nested PCR assay.

Optimizing real-time PCR method to detect Leptospira spp. in human blood and urine specimens.

Background: Leptospirosis is an acute infectious disease in humans caused by Leptospira spp. and classified as a zoonosis. Clinical symptoms of leptospirosis are nonspecific and the current available laboratory method for detecting Leptospira spp. is difficult, which resulted to the misdiagnosis of this disease. Therefore, the rapid and accurate method is needed to diagnose the disease. This study was aimed to optimize molecular diagnostic test using real-time PCR assay as a rapid, sensitive and specific method for the detection of pathogenic Leptospira spp. in humans. Methods: Bacterial DNA was extracted by DNA extraction kit according to the manufacturer’s instructions. Primers and probes used in this study was based on previous and published research. The assay is performed using PCR-IQTM5, iCycler Multicolor real-time PCR detection system. Specificity of the primer used was evaluated towards some bacterial pathogens. Results: Limit detection of the DNA was 0.375 fg/ml and the primers used does not cross-react with the genomes of the pathogens tested. Limit detection of DNA in blood is 150 fg/μl, and in urine is 1470 fg/μl. Conclusion: Real-time PCR test is a rapid and accurate method for detecting pathogenic Leptospira spp. in human specimens. Further research is needed to determine the sensitivity and specificity of real-time PCR tests compared with other diagnostic methods in clinical settings.

In vitro transcription of HIV-1 RNA for standard RNA.

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.

Simultaneous detection of Legionella species and Legionella pneumophila by duplex PCR (dPCR) assay in cooling tower water samples from Jakarta, Indonesia.

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.

A second generation of RT-PCR assay for detection of human immunodeficiency virus type 1 (HIV-1) infection.

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.

Development of multiplex-PCR assay for rapid detection of Candida spp.

Aim Candida spp. infection commonly occur in immunocompromised patients. Biochemical assay for identifi cation of Candida spp. is time-consuming and shows many undetermined results. Specifi c detection for antibody, antigen and metabolites of Candida spp. had low sensitivity and specifi city. In this study, we developed a rapid diagnostic method, Multiplex-PCR, to identify Candida spp. Methods Five Candida spp. isolates were cultured, identifi ed with germ tube and API® 20 C AUX (BioMerieux® SA) kit. Furthermore, DNA was purifi ed by QIAamp DNA mini (Qiagen®) kit for Multiplex-PCR assay. Results DNA detection limit by Multiplex-PCR assays for C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata were 4 pg, 0,98 pg, 0,98 pg, 0,5 pg and 16 pg respectively. This assay was also more sensitive than culture in that Multiplex-PCR could detect 2.6-2.9 x 100 CFU/ml, whereas culture 2.6-2.9 x 102 CFU/ml. Conclusion Multiplex-PCR is much more sensitive than culture and thus, can be recommended as a sensitive and specifi c assay for identifi cation of Candida spp.