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Isolation by the 5' anchored PCR technique and characterization of eighteen microsatellite loci in horseshoe crab (Tachypleus gigas)

Ling LP, Adibah AB, Tan SG, Christianus A, Faridah QZ

J Genet, 2011 Dec;90(3):e101-4.
PMID: 22232191
Matched MeSH terms: DNA Primers/chemistry
Molecular epidemiology of wild poliovirus type 1 in Europe, the Middle East, and the Indian subcontinent

Mulders MN, Lipskaya GY, van der Avoort HG, Koopmans MP, Kew OM, van Loon AM

J Infect Dis, 1995 Jun;171(6):1399-405.
PMID: 7769273

The genomic relationships of wild poliovirus type 1 strains recently isolated in Europe, the Middle East, and the Indian subcontinent was analyzed by automated amplicon sequencing of the VP1/2A junction region of the genome. Four major genotypes of poliovirus type 1 were found to circulate. Two genotypes were found predominantly in Eastern Europe, one of these in the Caucasian Region and the other in countries bordering the Black Sea. A third genotype circulated mainly in Egypt. The fourth and largest genotype circulated in the largest geographic area. Strains belonging to this genotype could be found in countries as far apart as Malaysia and Ukraine. Considerable genetic variation was observed among strains isolated in Egypt, Pakistan, and India, where poliovirus is endemic. Strains belonging to all four genotypes circulated in Pakistan. Data confirm the extent of poliovirus circulation in certain regions, stressing the need for intensification of vaccination in these regions.

Matched MeSH terms: DNA Primers/chemistry
Gold-nanoparticle based electrochemical DNA sensor for the detection of fish pathogen Aphanomyces invadans

Kuan GC, Sheng LP, Rijiravanich P, Marimuthu K, Ravichandran M, Yin LS, et al.

Talanta, 2013 Dec 15;117:312-7.
PMID: 24209346 DOI: 10.1016/j.talanta.2013.09.016

Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.

Matched MeSH terms: DNA Primers/chemistry
RB pocket domain B mutation frequency in Malaysia

Ishak SR, Hanafi H, Alagaratnam JV, Zilfalil BA, Tajudin LS

Ophthalmic Genet., 2010 Sep;31(3):159-61.
PMID: 20565234 DOI: 10.3109/13816810.2010.492816
Matched MeSH terms: DNA Primers/chemistry
Vairimorpha imperfecta n.sp., a microsporidian exhibiting an abortive octosporous sporogony in Plutella xylostella L. (Lepidoptera: Yponomeutidae)

Canning EU, Curry A, Cheney S, Lafranchi-Tristem NJ, Haque MA

Parasitology, 1999 Sep;119 ( Pt 3):273-86.
PMID: 10503253

The microsporidian genus Nosema is characterized by development in direct control with host cell cytoplasm, diplokaryotic nuclei throughout development and disporous sporogony. The genus Vairimorpha exhibits the same features plus an octoporous sporogony producing uninucleate spores in a sporophorous vesicle. A microsporidium from diamondback moth, Plutella xylostella, falls between Nosema and Vairimorpha in that it initiates but fails to complete the octosporous sequence in this host. The name Vairimorpha imperfecta n.sp. is proposed. Merogony is mainly by formation of buds from multinucleate meronts, the buds remaining attached in chains. Diplokaryotic spores measure 4.3 x 2.0 microns (fresh) and have 15.5 coils of the polar tube in 1 rank. The octosporous sporogony is aborted owing to irregular formation of nuclear spindles, incomplete cytoplasmic fission and bizarre deposition of electron-dense episporontal secretions. Phylogenetic analyses of the sequences of the small subunit rRNA genes of V. imperfecta and of several Nosema and Vairimorpha spp. place V. imperfecta in a clade with Nosema spp. from Lepidoptera rather than in the clade containing the more typical species of Vairimorpha. It is suggested that the ancestors of the Vairimorpha/Nosema complex of species exhibited both disporous and octosporous sporogonies, as does the type species of Vairimorpha, Vairimorpha necatrix. It would follow that true Nosema spp. have lost the ability to express an octosporous sequence and that V. imperfecta is in the process of losing it. It is proposed that the genera Nosema and Vairimorpha be placed in the same family Nosematidae Labbé 1899, rather than in separate families and orders as at present.

Matched MeSH terms: DNA Primers/chemistry
Cellular proteins bind to the 3' and 5' untranslated regions of dengue 2 virus genome

Ong CC, Lam SK, AbuBakar S

Malays J Pathol, 1998 Jun;20(1):11-7.
PMID: 10879258

In vitro generated cloned full length dengue 2 virus untranslated regions (UTRs) were used in RNA gel mobility shift assays to examine cellular factors binding to the virus genomes. Cellular factors in lysates of Vero (monkey) and C6/36 (mosquito) cells bound specifically and non-specifically to the dengue 2 virus 3' UTR. Non-specific interaction with the 5' UTR, resulting in formation of at least 4 band shift complexes was noted with lysate of the C6/36 cells only. Pre-treating the cell lysates with proteinase K affected binding of cellular factors to the dengue 2 virus UTRs, suggesting that the cellular factors were proteins. These findings suggest that cellular proteins could interact with specific sites on the dengue virus genomes.

Matched MeSH terms: DNA Primers/chemistry
Anion exchanger 1 mutations associated with distal renal tubular acidosis in the Thai population

Yenchitsomanus PT, Sawasdee N, Paemanee A, Keskanokwong T, Vasuvattakul S, Bejrachandra S, et al.

J Hum Genet, 2003;48(9):451-456.
PMID: 12938018 DOI: 10.1007/s10038-003-0059-6

We have previously demonstrated that compound heterozygous (SAO/G701D) and homozygous (G701D/G701D) mutations of the anion exchanger 1 (AE1) gene, encoding erythroid and kidney AE1 proteins, cause autosomal recessive distal renal tubular acidosis (AR dRTA) in Thai patients. It is thus of interest to examine the prevalence of these mutations in the Thai population. The SAO and G701D mutations were examined in 844 individuals from north, northeast, central, and south Thailand. Other reported mutations including R602H, DeltaV850, and A858D were also examined in some groups of subjects. The SAO mutation was common in the southern Thai population; its heterozygote frequency was 7/206 and estimated allele frequency 1.70%. However, this mutation was not observed in populations of three other regions of Thailand. In contrast, the G701D mutation was not found in the southern population but was observed in the northern, northeastern, and central populations, with heterozygote frequencies of 1/216, 3/205, and 1/217, and estimated allele frequencies of 0.23%, 0.73%, and 0.23%, respectively. The higher allele frequency of the G701D mutation in the northeastern Thai population corresponds to our previous finding that all Thai patients with AR dRTA attributable to homozygous G701D mutation originate from this population. This suggests that the G701D allele that is observed in this region might arise in northeastern Thailand. The presence of patients with compound heterozygous SAO/G701D in southern Thailand and Malaysia and their apparently absence in northeastern Thailand indicate that the G701D allele may have migrated to the southern peninsular region where SAO is common, resulting in pathogenic allelic interaction.

Matched MeSH terms: DNA Primers/chemistry
Genotypes of Japanese encephalitis virus isolated in three states in Malaysia

Tsuchie H, Oda K, Vythilingam I, Thayan R, Vijayamalar B, Sinniah M, et al.

Am J Trop Med Hyg, 1997 Feb;56(2):153-8.
PMID: 9080873

Two hundred forty nucleotides from the pre-membrane gene region of 12 Japanese encephalitis virus (JEV) strains isolated from three different regions of Malaysia from 1993 to 1994 were sequenced and compared with each other and with the JEV strains from different geographic areas in Asia. These 12 Malaysian isolates were classified into two genotypes. The four JEV strains isolated from Sarawak in 1994 and the four JEV strains isolated from Sepang, Selangor in 1993 were classified into one genotype that included earlier isolated strains from Malaysia (JE-827 from Sarawak in 1968 and WTP/70/22 from Kuala Lumpur in 1970). The four JEV strains from Ipoh, Perak in 1994 were classified into another genotype that included JEV strains isolated from northern Thailand and Cambodia. In an earlier report, 10 JEV strains from Sabak Bernam, Selangor in 1992 were classified into the largest genotype that included strains isolated in temperate regions such as Japan, China, and Taiwan. The data indicate that at least three genotypes of JEV have been circulating in Malaysia.

Matched MeSH terms: DNA Primers/chemistry
Low-density lipoprotein receptor gene mutations in a Southeast Asian population with familial hypercholesterolemia

Khoo KL, van Acker P, Defesche JC, Tan H, van de Kerkhof L, Heijnen-van Eijk SJ, et al.

Clin Genet, 2000 Aug;58(2):98-105.
PMID: 11005141 DOI: 10.1034/j.1399-0004.2000.580202.x

The aim of this study was to detect mutations in the genes coding for the low-density lipoprotein receptor and apolipoprotein B in patients of Southeast Asian origin with clinically diagnosed familial hypercholesterolemia (FH) and to relate these findings with the observed lower incidence of coronary heart disease in this part of the world. A total of 86 unrelated patients with FH were selected on clinical grounds, and complete DNA analysis of the low-density lipoprotein (LDL)-receptor and apolipoprotein B (apoB) genes by DGGE and DNA-sequencing was performed. In the majority (73%) of the cohort studied, no mutations could be detected, even after extensive analysis of the LDL-receptor and apoB genes. However, the 22 patients with a mutation had significantly more xanthomas and a higher incidence of coronary heart disease and levels of low-density lipoproteins were also significantly different. There was no correlation between the type of the mutation and lipoprotein levels or clinical signs of atherosclerosis. The fact that the majority of the FH patients studied had no detectable mutation and that this group had a significant milder phenotype, suggests the presence of a third gene in the Southeast Asian population, predominantly leading to a disorder resembling a milder form of FH. A similar, but less frequent, trait has recently been described in a number of European families.

Matched MeSH terms: DNA Primers/chemistry
Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay

Thong KL, Hoe SL, Puthucheary SD, Yasin R

BMC Infect Dis, 2005 Feb 14;5:8.
PMID: 15707504

In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay.

Matched MeSH terms: DNA Primers/chemistry
Molecular characterization of Salmonella weltevreden isolated from poultry: evidence of conjugal transfer of plasmid and antibiotic resistance

Radu S, Mutalib SA, Rusul G, Hassan Z, Yeang LK

Microbios, 2001;104(407):39-47.
PMID: 11229656

Ten strains of Salmonella weltevreden isolated from poultry sources were examined and found to contain plasmid DNA ranging in size from 1.8 to 68.5 MD. All isolates were susceptible to carbenicillin, cephalothin, ceftriazone, gentamicin, kanamycin and nalidixic acid, but resistance to bacitracin (100%), penicillin G (100%), rifampicin (100%), sulphamethoxazole (100%), cefuroxime (80%) and tetracycline (60%) was recorded. The 55 MD plasmid of strain SW5 determined resistance to penicillin G and tetracycline, which was transmissible to the E. coli K12 recipient at a frequency of 3.52 x 10(-5) transconjugants per input donor cell. The results of arbitrarily primed polymerase chain reaction (AP-PCR), using two 10-mer oligonucleotides and PCR-ribotyping to differentiate between the ten strains of S. weltevreden were compared. The strains were separated into ten different genome types by AP-PCR but were indistinguishable by PCR-ribotyping. These results suggest that poultry may constitute a reservoir for disseminating antibiotic resistance and that AP-PCR may be a valuable tool for epidemiological studies.

Matched MeSH terms: DNA Primers/chemistry
Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations

Tang K, Ngoi SM, Gwee PC, Chua JM, Lee EJ, Chong SS, et al.

Pharmacogenetics, 2002 Aug;12(6):437-50.
PMID: 12172212

The MDR1 multidrug transporter plays a key role in determining drug bioavailability, and differences in drug response exist amongst different ethnic groups. Numerous studies have identified an association between the MDR1 single nucleotide polymorphism (SNP) exon 26 3435C>T and differences in MDR1 function. We performed a haplotype analysis of the MDR1 gene in three major ethnic groups (Chinese, Malays and Indians) by examining 10 intragenic SNPs. Four were polymorphic in all three ethnic groups: one occurring in the non-coding region and three occurring in coding exons. All three coding SNPs (exon 12 1236C>T, exon 21 2677G>T/A and exon 26 3435C>T) were present in high frequency in each ethnic group, and the derived haplotype profiles exhibited distinct differences between the groups. Fewer haplotypes were observed in the Malays (n = 6) compared to the Chinese (n = 10) and Indians (n = 9). Three major haplotypes (> 10% frequency) were observed in the Malays and Chinese; of these, two were observed in the Indians. Strong linkage disequilibrium (LD) was detected between the three SNPs in all three ethnic groups. The strongest LD was present in the Chinese, followed by Indians and Malays, with the corresponding LD blocks estimated to be approximately 80 kb, 60 kb and 40 kb, respectively. These data strongly support the hypothesis that strong LD between the neutral SNP exon 26 3435C>T and a nearby unobserved causal SNP underlies the observed associations between the neutral SNP and MDR1 functional differences. Furthermore, strong LD between exon 26 3435T and different unobserved causal SNPs in different study populations may provide a plausible explanation for conflicting reports associating the same exon 26 3435T allele with different MDR1 functional changes.

Matched MeSH terms: DNA Primers/chemistry
Type-3 dengue viruses responsible for the dengue epidemic in Malaysia during 1993-1994

Kobayashi N, Thayan R, Sugimoto C, Oda K, Saat Z, Vijayamalar B, et al.

Am J Trop Med Hyg, 1999 Jun;60(6):904-9.
PMID: 10403318

To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.

Matched MeSH terms: DNA Primers/chemistry
Improvement in the detection rate of t(14;18) translocation on paraffin-embedded tissue: a combination approach using PCR and FISH

Shaminie J, Peh SC, Tan MJ

Pathology, 2003 Oct;35(5):414-21.
PMID: 14555386

AIMS: PCR has been the primary method used for the detection of t(14;18) translocation in formalin-fixed, paraffin-embedded tissues. This technique mainly targets the well-characterised breakpoint regions in chromosomes 14 and 18. FISH is now applicable on paraffin tissue sections and has been suggested to be capable of detecting essentially 100% of t(14;18) translocated cases. In this study, we described the application of both PCR and FISH for the detection of t(14;18) translocation.
METHODS: Fifty follicular lymphoma cases were retrieved from the files of the Department of Pathology, University of Malaya Medical Centre (UMMC). Nested PCR amplification of MBR/JH and mcr/JH was performed in these cases, and those cases that did not demonstrate the translocation were subjected to FISH analysis.
RESULTS: Thirty cases (60%) had t(14;18) translocation detected by PCR, 25 (50%) had breakpoint with MBR and five (10%) involved mcr. Twenty cases without detectable t(14;18) translocation by PCR were analysed by FISH. Eleven cases were successfully probed, and four of them showed positive translocation signal.
CONCLUSIONS: The combination of PCR and FISH analysis on paraffin tissue sections for the detection of t(14;18) translocation increases the sensitivity of detection from 60 to 68%. Problems encountered in our FISH analysis on tissue sections impose certain limitations in using this technique for retrospective screening of large number of samples. Therefore, we suggested the application of PCR as the first screening tool on retrospective archival materials, followed by FISH on those PCR-negative cases.

Matched MeSH terms: DNA Primers/chemistry
Analysis of fliC variation among clinical isolates of Burkholderia cepacia

Winstanley C, Hales BA, Morgan JAW, Gallagher MJ, Puthucheary SD, CISSé MF, et al.

J Med Microbiol, 1999 Jul;48(7):657-662.
PMID: 10403416 DOI: 10.1099/00222615-48-7-657

PCR and restriction fragment length polymorphism (RFLP) typing of flagellin genes (fliC) from 57 clinical isolates of Burkholderia cepacia indicated that only type 11 flagellins were present. Twenty-two isolates previously identified as the epidemic UK cystic fibrosis strain were indistinguishable by this method, as were 11 isolates from a pseudo-outbreak in Senegal. Other clinical isolates, including 19 from disparate sources in Malaysia, were separated into nine fliC RFLP groups, exhibiting a large degree of divergence. When isolates were indistinguishable by fliC genotyping, their similarity was confirmed by whole genome macro-restriction analysis with pulsed-field gel electrophoresis following XbaI digestion. The variation in fliC sequences of B. cepacia was far greater than that with B. pseudomallei, supporting the view that 'B. cepacia', as currently defined, may comprise several different genomic species.

Matched MeSH terms: DNA Primers/chemistry
Detection of Chlamydia trachomatis in urine samples by polymerase chain reaction and enzyme immunoassay

Ngeow YF, Hema V, Zakaria M, Lee CH, Ramachandran S

Malays J Pathol, 1997 Dec;19(2):127-32.
PMID: 10879253

First-void urine samples collected from sexually transmitted diseases (STD) clinic patients were examined by a nested polymerase chain reaction (PCR) and a commercial enzyme immunoassay (IDEIA Chlamydia) for the diagnosis of Chlamydia trachomatis urethritis or cervicitis. The primers for the PCR amplified a target in the major outer membrane protein (MOMP) gene in C trachomatis while the IDEIA detected genus-specific chlamydial lipopolysaccharide. Discrepant results were resolved by retesting urine specimens with a second (plasmid-based) PCR and taking urethral or endocervical swab results into consideration. For 231 men (chlamydial prevalence 20.4%), the sensitivity, specificity, positive and negative predictive values were 59.6%, 99.5%, 96.6% and 90.6% for urine IDEIA, 68.1%, 99.5%, 97% and 92.4% for urethral swab IDEIA and 97.9%, 99.5%, 97.9% and 99.5% for urine PCR. The corresponding rates for 66 women (chlamydial prevalence 54.6%) were 19.4%, 100%, 100% and 50.8% for urine IDEIA, 86.1%, 96.7%, 96.9% and 85.3% for endocervical swab IDEIA and 91.7%, 93.3%, 94.3% and 90.3% for urine PCR. Hence, in a high prevalence population, the urine IDEIA was a suitable alternative to the male urethral swab IDEIA but significantly less sensitive than the endocervical swab IDEIA. The urine PCR was, however, much more sensitive than the urine IDEIA for both men and women and could replace the endocervical swab IDEIA for the diagnosis of chlamydial cervicitis.

Matched MeSH terms: DNA Primers/chemistry
Alterations of the p14ARF-p53-MDM2 pathway in oral squamous cell carcinoma: MDM2 overexpression is a common event

Lim KP, Sharifah H, Lau SH, Teo SH, Cheong SC

Oncol Rep, 2005 Oct;14(4):963-8.
PMID: 16142358 DOI: 10.3892/or.14.4.963

The majority of global incidences of oral cancer occur in Asia, and the aetiology of oral cancer is different in Asia as it is in the West. However, whereas there is a growing understanding of the molecular mechanisms of oral cancer progression in the West, there is little progress in this understanding in Asia. In particular, the role of the p53 pathway in modulating cancer progression in Asian oral cancer remains unclear. In this study, we micro-dissected and analysed 20 well-differentiated oral squamous cell carcinoma specimens for alterations in the p53 pathway. We found that 6/20 samples contained mutations in the p53 gene which occurred in three hotspots, at codon 203, 218 and 296. Furthermore, 6/20 samples had a homozygous deletion of p14ARF, but notably p14ARF deletion and p53 mutation events were often independent and mutually exclusive. Strikingly, MDM2 was upregulated in 20/20 samples, but not in 3/3 normal tissue specimens. Taken together, these data suggest that inactivation of the p53 pathway is a frequent event in oral squamous cell carcinoma, which occurs by an aberration in one of a number of players in the p53 pathway.

Matched MeSH terms: DNA Primers/chemistry

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