Welcome to American Journal of Microbiological Research

American Journal of Microbiological Research is a peer-reviewed, open access journal that provides rapid publication of articles in all areas of microbiological research. The goal of this journal is to provide a platform for scientists and academicians all over the world to promote, share, and discuss various new issues and developments in different areas of microbiological research.

ISSN (Print): 2328-4129

ISSN (Online): 2328-4137

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Website: http://www.sciepub.com/journal/AJMR

   

Article

The Evaluation on Molecular Techniques of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP), Reverse Transcription Polymerase Chain Reaction (RT-PCR), and Their Diagnostic Results on MinIONTM Nanopore Sequencer for the Detection of Dengue Virus Serotypes

1Department of Clinical Pathology, Faculty of Medicine, Sam Ratulagi University, Manado, Indonesia

2Department of Physiology, Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia

3Department of Parasitology, Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia.

4Molecular Biology and Immunology Laboratory, Department of Microbiology, Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia


American Journal of Microbiological Research. 2015, 3(3), 118-124
doi: 10.12691/ajmr-3-3-4
Copyright © 2015 Science and Education Publishing

Cite this paper:
Arthur Elia Mongan, Irawan Yusuf, Isra Wahid, Mochammad Hatta. The Evaluation on Molecular Techniques of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP), Reverse Transcription Polymerase Chain Reaction (RT-PCR), and Their Diagnostic Results on MinIONTM Nanopore Sequencer for the Detection of Dengue Virus Serotypes. American Journal of Microbiological Research. 2015; 3(3):118-124. doi: 10.12691/ajmr-3-3-4.

Correspondence to: Mochammad  Hatta, Molecular Biology and Immunology Laboratory, Department of Microbiology, Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia. Email: hattaram@indosat.net.id

Abstract

Background and aims:. Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay is a method described as a simple, rapid, and cost-effective, and a powerful gene amplification technique for rapid identification of microbial infection. The aims of this study were to validate a reverse-transcription LAMP method (RT-LAMP) with a reverse-transcription PCR technique (RT-PCR) for DENV serotype detection, and to evaluate the potential of this RT-LAMP method from which its amplification product can be applied for MinIONTM Nanopore sequencing. Methods: This study was performed by using 26 patient serum samples from the Confirmed group and 23 sera from the Suspected group, and 10 sera from Healthy volunteer group. The same validation study to confirm the sensitivity and specificity between the RT-LAMP assay and RT-PCR was also conducted by making a serial dilution of template as well as dengue serotype detected sera. Furthermore, experiments of MinIONTM Nanopore sequencer using the amplification product (amplicon) of RT-LAMP assay from patient serum samples and its comparative diagnostics with RT-PCR and Sanger’s Sequencing were also done. Results: From the validation study, 17 (65.4%) out of the 26 sera of Confirmed group were serotype detected by RT-LAMP assay, compared to that of RT-PCR which was only 10 (38.4%). Same result in Suspected group, there were 11 (47.8%) out of 23 sera of Suspected group were serotype detected by RT-LAMP assay, compared to that of RT-PCR which was only 1 (4.3%). For the Healthy group, the result was somewhat comparable between the two methods. In the serial dilution study, RT-LAMP was superior than that of the RT-PCR technique, particularly seen in the serial dilution of clinical serum samples. The limit of detection of RT-LAMP was down to the titer of 1/1000x, while RT-PCR was only 1/10x. Secondly, amplicon of RT-LAMP assay from patient serum samples fits to be sequenced by the MinIONTM. In the comparative study, the results showed that RT-LAMP was more sensitive and specific compared to that of the RT-PCR technique and Sanger’s Sequencing. Conclusions: RT-LAMP assay was more sensitive to that of RT-PCR technique. In the comparative study using MinIONTM Nanopore sequencer for the detection of dengue virus serotypes, the RT-LAMP was more sensitive and specific to that of RT-PCR and Sanger’s Sequencing. The method of RT-LAMP should be applied more because its sensitive and specific, simple, rapid, cost-effective, and also can be a tool of point of care testing for the detection of dengue virus serotypes.

Keywords

References

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Article

Genotyping of Pulmonary Mycobacterium tuberculosis Isolates from Sudan Using Spoligotyping

1Tropical Medicine Research Institute, National Center for Research, Khartoum, Sudan

2College of Applied Medical Science, Shaqra University, Shaqra, KSA

3Blood Transfusion Service, National Blood Transfusion Center, Khartoum, Sudan

4Tuberculosis Research Center, University of Kassala, Kassala, Sudan

5National Tuberculosis Reference Laboratory, National Laboratory of Public Health, Khartoum, Sudan

66Molecular biology Armauer Hansen Research Institute, Addis Ababa, Ethiopia

7Faculty of Science and Technology, Omdurman Islamic University, Khartoum, Sudan

88Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan


American Journal of Microbiological Research. 2015, 3(4), 125-128
doi: 10.12691/ajmr-3-4-1
Copyright © 2015 Science and Education Publishing

Cite this paper:
Muataz M. Eldirdery, Intisar E. Alrayah, Mona OA. Awad ElkareIm, Fatima A. Khalid, Asrar M A/Salam Elegail, Nuha Y. Ibrahim, Eman O M. Nour, Rahma H. Ali, Elena Hailu, Markos Abebe, Abraham Aseffa, Najem Aldin M. Osman, Maowia M. Mukhtar, Nihad M A. Elhaj, Atif A. Elagib. Genotyping of Pulmonary Mycobacterium tuberculosis Isolates from Sudan Using Spoligotyping. American Journal of Microbiological Research. 2015; 3(4):125-128. doi: 10.12691/ajmr-3-4-1.

Correspondence to: Muataz  M. Eldirdery, Tropical Medicine Research Institute, National Center for Research, Khartoum, Sudan. Email: eldirdery5@hotmail.com

Abstract

Tuberculosis (TB) remains a major public health problem worldwide due to its high risk of person-to-person transmission, morbidity and mortality [1]. Sudan has a high burden of tuberculosis. Spoligotyping (spacer oligonucleotide typing) a rapid method for genotyping of Mycobacterium tuberculosis using the principle of reverse hybridization. The ecology of the prevalent mycobacteria strain can vary depending on country and region. The aim of this study was to determine the genotyping of Mycobacterium tuberculosis isolated from Sudan using spoligotyping SPOLDB4. A total of 75 Mycobacterium tuberculosis sputum samples were collected from pulmonary Tuberculosis patients attending references Laboratories and diagnostic centers in Khartoum and Eastern Sudan in (2011-2013). The mycobacteria were genotyped using Spoligotyping technique and data obtained were analyzed and compared to the SPOLDB4 database. Among the 75 isolate analyzed, 57(76%) were identified by SPOLDB4 and 18 (24%) could not be matched to any lineages. The most prevalent genotype cluster was MANU2 38 (50.7%) followed by CASI Delhi 8 (10.7%). In the study SIT54 was the most common pattern 37 (49.3%) followed by SIT25 6(8%).

Keywords

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Article

Immunomodulator Potential of Miana Leaves(Coleus scutellarioides (L) Benth) in Prevention of Tuberculosis Infection

1Health Technology Department, Ministry of Health, Makasssar, Indonesia

2Faculty of Public Health, Airlangga University, Surabaya, Indonesia

3Faculty of Life Sciences and Technology, Airlangga University, Surabaya, Indonesia

4Department of Microbiology, Faculty of Medicine, Tadulako University, Palu, Indonesia

5Molecular Biology and Immunology Laboratory for Infectious Diseases, Microbiology Department, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia

6Department of Microbiology and Immunology, Faculty of Medicine, Mulawarman University, Samarinda, Indonesia


American Journal of Microbiological Research. 2015, 3(4), 129-134
doi: 10.12691/ajmr-3-4-2
Copyright © 2015 Science and Education Publishing

Cite this paper:
Sesilia Rante Pakadang, Chatarina Umbul Wahjuni, Hari Basuki Notobroto, Dwi Winarni, Ressy Dwiyanti, Yadi, Muhammad Sabir, Mochammad Hatta. Immunomodulator Potential of Miana Leaves(Coleus scutellarioides (L) Benth) in Prevention of Tuberculosis Infection. American Journal of Microbiological Research. 2015; 3(4):129-134. doi: 10.12691/ajmr-3-4-2.

Correspondence to: Mochammad  Hatta, Molecular Biology and Immunology Laboratory for Infectious Diseases, Microbiology Department, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia. Email: hattaram@indosat.net.id

Abstract

Aim, The aim of this study to investigate the immunonomodulator effects of miana leaves (Coleus scutellarioides (L) Benth) on prevention of tuberculosis in wistar rats, Method, Samples of white male Wistar rats were divided into 4 groups, the samples were treated with miana leaves extract (EDM) and then infected by intra tracheal M. tuberculosis H37Rv strain and subsequently given a placebo, EDM and GAB (combined Rifampicin and EDM) and healthy control animals were treated with EDM. In this study we measure the level of Immunomodulatory parameter; the number of T-lymphocytes and CD4 T-cells measured by flowcytometry method, the levels of IFN-γ and TNF-α were measured by ELISA and the number of M. tuberculosis colonies derived from the rat lung in LJ media. Result, Results showed the increasing of T-lymphocytes, CD4 T-Cells, the levels of IFN-γ, TNF-α and decreasing in the number of M. tuberculosis colonies after EDM treatment. Conclusion, Miana leaves extract (Coleus scutellarioides, (L) Benth.) increased the number of T-lymphocytes, CD4 T-cell counts, levels of IFN-γ and TNF-α and decreased in the number of M. tuberculosis colony in infected wistar lung.

Keywords

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