Molecular approach for rapid diagnostic kit to detect Yersinia pestis from infected soil, water sample

Yersinia pestis is the cause for the acute infection and may be chosen for biological terrorism. Rapid diagnosis of this agent from infected soil - water is essential. Y. pestis habours 3 specific plasmids providing virulent factors to the bacterium. Objectives: (1) Testing the sensitivity and accurateness of PCR for Y. pestis. (2) Carrying out PCR using total genomic DNA serially diluted as a template. (3) Undertaking PCR on artificical experimentation by diluting Y. pestis in soil water as samples to test PCR based fast diagnostic approach. Methods: Yersinia pestis (inactivated) was used. Genomic DNA was extracted by DNeasy kit (Qiagen Inc). Using primer - pairs PLAF - PLAR (binding on pia gene of plasmid pPCP1) a specific product of PCR was 480 bp. After determination of the PCR sensitivity, a molecular based diagnostic kit was developed. Sensitivity and specificity of this kit was tested by PCR using diluted genomic DNA and bacterium itself; and mix of these templates in water and soil as samples. Results: With the diluted genomic DNA, it was successful to obtain specific PCR with 0.6ng template, which is equal to a single bacterium. Additionally, successful PCR amplification was obtained using the whole bacterium (without extraction of genomic DNA) and diluted quantity ranging from 101 to 102. Based on these results, the bacterium was artificially diluted with sample of soil - water as a natural isolate for PCR amplification. Conclusions: Evidently, approach for PCR-based diagnostic kit was successfully carried out from any template including soil - water samples with high fidelity, using the pia gene genetic marker of pPCR of Y. pestis.

Molecular identification of Paragonimus heterotremus from different hosts in Vietnam using mitochondrial genetic markers

By molecular identification and using mitochondrial genetic markers, the results showed that different forms of lung fluke in Vietnam, including adult fluke from human, dogs, cats and Potamicus sp. rock crab, have been identified as Paragonimus heteotremus. Molecular-based analysis on 390 nucleotides of the cytochrome oxidase gene revealed that Paragonimus heterotremus of Vietnam from all forms showed high identity to the Chinese and Thai strains. (99.0 -99.2% nucleotide and 97-100% amino acid). Phylogenetic analysis uniquely placed the Vietnamese Paragonimus sp to the group of P.heterotremus of Chinese and Thai origin. Thus, P.heterotremus is offically identified from most of the natural and experimental hosts in Vietnam

First report on molecular identification of opisthorchis viverrini isolated in An My (Tuy An, Phu Yen)

A region of 326 bp of mitochondrial cytochrome oxidase 1 (cox1) for Opisthorchis sp sample collected in An My, Tuy An, Phu Yen, was amplified using PCR. Nucleotide sequence of this cox1 fragment was used to comparatively analyzed with that of O. viverrini, strain Khon Kaen, Thailand, and C. sinensis sequences of Opisthorchis sp of Vietnam, China and Korea. The analysis revealed that nucleotide sequences of Opisthorchis sp of Vietnam has absolute homology to O. viverrini strain Khon Kaen, Thai Lan, but difference from C. sinensis originated from Vietnam, China and Korea. Opisthorchis sp of Vietnam isolated in Phu Yen is, thus, molecularly identified as Opisthorchis viverrini. This gives rise to establish a diagnostic approach and aspect of preventive/control for these species

Molecular identification and phylogenetic analysis of human parasitic Taenia sp samples isolated in Vietnam

A portion of 652bp of mitochondrial-encoded cob gene was amplified by polymerase chain reaction (PCR) from different Taenia sp samples (tspVN1-10) of different forms of adult, cysticercus isolated in Vietnam. The nucleotide and amino acid sequences of Vietnamese Taenia sp samples were comparatively aligned with the known corresponding sequences of other Taenia species in GenBank. Results showed that TspVN1-3 is Taenia asiatica, TspVn4-6 is Taenia saginata, and TspVN7-10 is Taenia solium. The Vietnamese T.solium is clustered with the Asian T.solium species, while the Vietnamese T.asiatica is clustered with Taiwanese and T.saginata together with the Chinese T.saginata isolate

18S ribosomal RAN besed molecular identification of giant intestinal fluke (fasciolopsis buski) isolated in human in Vietnam

The total sequence of 18S rRNA and the neighbours including 1950 pairs of nucleotide (N) was received by PCR and expressed orderly. The examinated sample was 1 of 8 adult worms collected from 12.5 years old male patient. The worm was determined morphologically as Fasciolopsis buski. The comparison showed that in this intestinal fluke worm there is an almost absolutely analogous coefficient concerning nitrogen components of 18S rRNA in only 2 differences on 1950 N (0.01%) with the gene sequence of 18S rRNA kept in the bank of genes. In Viet Nam, this is the first molecular determination realized on human

Identification for Mycobacterium leprae from patients in Vietnam with molecular biology method

Using polymerase chain reaction (PCR) and sequencing, 492 nucleotides of pra gene was obtained from clinical swabs of the Vietnamese patients for comparative analysis with the corresponding sequences of the global strains of Mecobacterium leprae. As entry data for searching in Genbank, the Vietnamese strains of M.leprae showed very high homology level to the other global strains. In comparision to the standard TN strain, absolute homology (100%) for QH1 (designated as MLVN1) and 98-99% homology for QH2 strain (designated as MLVN2) of the Vietnamese M. leprae was observrd. There may be a number of variants among the M. leprae population in Vietnam. For final conclusion, thus, investigation must be done for many other isolates of patients and geographic origins. This is the first molecular-based identification for M. leprae in Vietnam. Our results afford establishing diagnostic and identification methodologies/techniques for M. leprae from clinical swabs from patients in Vietnam

Molecular identification of the Vietnamese yersinia pestis isolated in Vietnam using pla sequence of plasmid pPCP1 as genetic marker

Pla sequence composes of 480 nucleodides of pPCP1 plasmide originated from Yersinia pestis isolated from Viet Nam and collected by the technique of PCR of chemical line into TA vector and demonstrated in turn. PCR reaction gave specific result from 3 diversified sources for mould: total extracted ADN, ADN extracted from plasmide isolated from bacterium complete cell. A fast and accurate method of diagnosis was created basing on this operation. By BLAST programme for accessing Gene Bank, the pla-gene sequence of Yersinia pestis in Viet Nam is analogue to other strains worldwide