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PubMed2004

[Molecular epidemiological analysis by IS1245-based restriction fragment length polymorphism typing on cases with pulmonary Mycobacterium avium disease observed in the same family].

INTRODUCTION: Mycobacterium avium-intracellulare complex (MAC) has become one of major human pathogens, however, its routes of transmission and environmental reservoirs causing human infection were not yet elucidated. We reported three families affected by pulmonary Mycobacterium avium (M. avium) disease. Previous reports on MAC diseases observed in the same family were very rare. The purposes of this study were to investigate whether the infected M. avium was the same strain among cases in the same family and to examine the possibility of human-to-human transmission, or infection from exposure to a common environmental reservoir. METHODS: M. avium isolates from nine cases of three families were examined by DNA polymorphism based typing technique, restriction fragment length polymorphism (RFLP) analysis using insertion sequence IS1245 as a probe, to type the strains. Some isolates were subcultured to a single clone. RESULTS: All strains isolated from cases in the same family showed different patterns by the RFLP analysis. And not only simultaneous polyclonal infection but also repeated polyclonal infections were observed in some patients. DISCUSSION: The results suggest importance of underlying anti-mycobacterial immunological impairment and defects of local defense rather than virulence of infected strains as the pathogenesis of pulmonary M. avium disease.

Aged↗
PubMed1995

Restriction fragment length polymorphism (RFLP) analysis of genomic DNA of 5 strains of Trichinella spiralis in China.

Five restriction endonucleases were used to digest genomic DNA from 5 isolates of Trichinella spiralis obtained from Changchun, Tianjin, Xian, Henan and Yunnan. All the isolates were secured from pigs except the Changchun strain which came from dog. The DNA fragments digested by endonuclease were separated by agarose gel electrophoresis. The Changchun isolate had a EcoRI band at 1.12kb and a DraI band at 1.97kb which were unique to this isolate. A cloned specific repetitive DNA sequence (1.12kb) from the Changchun strain was selected to prepare a probe for the Southern blotting of EcoRI restriction DNA fragments for the 5 isolates. The 1.12kb hybridizing band did not appear except in the Changchun isolate. These results seem to indicate that there are differences between the isolates obtained from hosts in different geographical regions.

Animals↗
PubMed2002

Polymerase chain reaction based restriction fragment length polymorphism for the genotyping of cytomegalovirus (CMV) from patients with CMV disease in Chennai.

BACKGROUND & OBJECTIVES: Since cytomegalovirus (CMV) is heterogenous and exhibits genomic polymorphism, polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) was applied to identify the glycoprotein B subtypes in patients diagnosed to have CMV disease. METHODS: CMV standard strain (AD 169) and 55 clinical specimens from 50 patients (35 males; 15 females) positive for CMV by PCR coding for the morphological transforming region II gene were subjected to PCR coding for gp 55 region of CMV gB gene. PCR amplified products were subjected to RFLP using Hinf I. RESULTS: Of 50 patients, 26 (52.0%) (20 males; 6 females) were positive by PCR (gB gene). Upon RFLP, AD 169 and CMV strains of 14 (53.8%) patients (14 males) were typed as subtype II, and 12 (46.1%) (6 males; 6 females) as subtype III. Of 11 paediatric patients, 7 (63.6%) were infected with CMV subtype II and 4 (36.4%) with subtype III. CMV strains of the dual specimens from 3 PCR positive patients belonged to the same subtype. INTERPRETATION & CONCLUSION: In this study on CMV genotypes, CMV gB subtypes II and III were found in 53.8 and 46.1 per cent patients studied respectively, while I and IV were not present.

Adult↗
PubMed1991

Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis.

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype.

Animals↗
PubMed2000

Rapid detection of dihydropteroate polymorphism in AIDS-related Pneumocystis carinii pneumonia by restriction fragment length polymorphism.

Sulpha agents, which act by inhibiting the enzyme dihydropteroate synthase (DHPS), are used widely for the treatment and prophylaxis of Pneumocystis carinii pneumonia (PCP). Recently, we have shown that mutations in the dihydropteroate synthase (DHPS) gene of Pneumocystis carinii f.sp hominis are associated with failure of sulpha prophylaxis and increased mortality in HIV-1 positive patients with PCP, suggesting that DHPS mutations may cause sulpha resistance. To facilitate detection of DHPS mutations we developed a restriction fragment length polymorphism (RFLP) assay, detecting mutations at codon 55 and 57 of the P. carinii DHPS gene. The RFLP-assay was compared with direct DNA sequencing on 27 PCP isolates from HIV-1 positive patients with a mixture of wildtype and mutant DHPS types. In all samples the RFLP-assay correctly identified wildtype or DHPS mutation at codon 55 or 57. Combined with DNA extraction by a Chelex-based method, this method can be performed within 1 d and allows a fast, cost-efficient and reliable method of detection of DHPS mutations in P. carinii.

AIDS-Related Opportunistic Infections↗
PubMed1993

Polymerase chain reaction-based restriction fragment length polymorphism analysis of a fragment of the ribosomal operon from Rochalimaea species for subtyping.

Restriction endonuclease analysis of a polymerase chain reaction-amplified DNA fragment which included the spacer region between the genes coding for 16S and 23S rRNAs and a portion of the gene coding for 23S rRNA (spacer + 23S) was done on 10 previously characterized clinical isolates of Rochalimaea henselae, one clinical isolate of Rochalimaea quintana, and the type strains of R. henselae, R. quintana, Rochalimaea vinsonii, and Bartonella bacilliformis. Brucella abortus DNA was not amplified by the primer set used. The clinical isolates of Rochalimaea were obtained from blood or tissue from patients with and without preexisting disease. The amplicon from each strain was digested with five endonucleases (AluI, HaeIII, TaqI, HinfI, and MseI). AluI and HaeIII were useful in species differentiation and subtyping of R. henselae. R. henselae isolates showed six different restriction patterns with AluI and four patterns with HaeIII. TaqI, HinfI, and MseI were useful only in species differentiation. These observations indicate that PCR amplification of the spacer + 23S region of the ribosomal DNA of Rochalimaea spp., along with restriction endonuclease analysis, allows differentiation of Rochalimaea spp. from closely related genera, differentiation among the species within Rochalimaea, and differentiation of strains within R. henselae. The subtyping potential of this method may be useful for further clinical and epidemiologic studies of the spectrum of diseases caused by R. henselae.

Bacterial Typing Techniques↗
PubMed2001

recA genotyping of Salmonella enteritidis phage type 4 isolates by restriction fragment length polymorphism analysis.

AIMS: To subtype Salmonella enteritidis phage type 4 isolates by using recA genotyping. METHODS AND RESULTS: Random amplified polymorphic DNA analysis using a primer ERIC2 of 76 isolates of Salmonella enteritidis phage type 4 obtained in Northern Ireland in 1998 and in 1999 demonstrated the presence of five genotypes. Restriction fragment length polymorphism analysis, using a degenerate primer pair designed to amplify a segment (about 640 bp in length) of the recA gene from several members of the Enterobacteriaceae with restriction enzymes, HhaI and Sau3AI, showed that the resulting fragments could differentiate the isolates into three groups, respectively. CONCLUSION: recA gene amplification and HhaI and Sau3AI restriction digestion was demonstrated to increase the differentiating power between isolates of Salmonella enteritidis phage type 4 by combining the patterns of the random amplified polymorphic DNA analysis procedure using a primer ERIC2. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel restriction fragment length polymorphism assay for isolates of Salmonella enteritidis phage type 4, based on the amplification of the recA gene was attained and its comparison and its combination with random amplified polymorphic DNA analysis was provided.

Bacterial Proteins↗
PubMed1991

[DNA polymorphism in the Mongolian population. Restriction fragment length polymorphism analysis of DNA at seven loci of the nuclear genome].

Seven DNA markers from five genes and one chromosomal region were analysed in Mongolian population using the polymerase chain reaction. The frequencies of alleles of the polymorphisms detected with HindIII in the HBG-2, AvaII in the HBB, MspI and XbaI in the Apo-B, PstI in the D7S8, HincII in the LDLR and allele frequency of the minisatellite fragment in the AT-3 have been determined. The results of the RELP for Apo-B(MspI), LDLR, D7S8 and AT-3 are obtained for the first time among Mongoloids. DNA markers studied demonstrated high level of polymorphisms in the population of Mongolia, except for XbaI and MspI restriction sites at the Apo-B locus. The data obtained for Mongolian population and the literature data were compared.

Alleles↗
PubMed2006

A novel intronic mutation that may affect genotyping result of CYP2C8 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is strongly associated with CYP2C8*3 in a South American population.

During the investigation of a CYP2C8*3 genetic polymorphism in a South American population, we obtained a discrepant result using two different polymerase chain reaction (PCR) protocols. A single nucleotide polymorphism (SNP) (IVS3+43 G>C) was identified in the intron 3 region, which was used as a primer-annealing site of one of the two PCR protocols. A genotyping method was developed to enable discrimination of the CYP2C8*1A, CYP2C8*3 (416 G>A), and CYP2C8*3 (416G>A; IVS3+43 G>C) alleles. In a screen of a South American population, we found that individuals carrying the CYP2C8*3 (416 G>A) polymorphism also carried the CYP2C8*3 (416G>A; IVS3+43 G>C). However, we did not find any carriers of CYP2C8*3 (416G>A; IVS3+43 G>C) in a Japanese population.

Aryl Hydrocarbon Hydroxylases↗
PubMed1993

Genetic structure of natural populations of Dryas iulia (Lepidoptera: Nymphalidae) revealed by enzyme polymorphism and mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP).

Dryas iulia appears to have undergone a mode of evolution different from that of other members of its subfamily (Heliconiinae). While other species constitute highly subdivided and inbred populations, those of D. iulia are thought to be large and uniform. Analyzing six samples from Southern Brazil (state of Rio Grande do Sul) in relation to three enzyme systems (EST, LAP, and PGM) and their mtDNA RFLP patterns, we found that they are very similar at the molecular level. The F statistics for enzyme polymorphism data revealed that inbreeding makes a great contribution to the population homozygosity, since FIS equals 0.1322 and FST equals 0.0023. Since the chi-square test showed that FST is not significant, we conclude that all localities belong to the same population. The mtDNA differentiation was about 12 times greater than for nuclear genes; FST was equivalent to 0.0265. We suggest that this difference is due to a higher dispersal of males, in relation to females.

Animals↗
PubMed1987

Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQw1 and DQw3 alleles of the HLA-D region.

Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQ beta, DQ alpha, and DR beta cDNA probes. Hybridization with the DQ beta probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQ alpha and DR beta probes failed to identify disease-specific restriction fragment length polymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment length polymorphism that is highly associated with susceptibility to autoimmune disease.

Alleles↗
PubMed1988

HLA-DP typing by analysis of DNA restriction fragment length polymorphisms in the HLA-DP beta subregion.

HLA class II antigens are encoded in the HLA-D region and are highly polymorphic. Southern hybridization technique was used to analyze restriction fragment length polymorphisms (RFLPs) in the DP beta gene and an attempt was made to correlate these with DP haplotypes derived from primed lymphocyte typing (PLT) analysis. Digestion of DNA from 32 Epstein-Barr virus (EBV)-transformed cell lines (of haplotypes DPw2, DPw3, DPw4, DPw5, and Cp63) with three different restriction endonucleases. Southern transfer, and hybridization to the DP beta cDNA probe revealed multiple fragments in all cell lines tested. The polymorphic patterns of these fragments were found to correlate with DP haplotypes, suggesting the possibility that the analysis of DNA RFLPs (DNA typing) in the HLA-DP beta subregion can distinguish and identify HLA-DP haplotypes.

DNA↗
PubMed1991

Genomic analysis of a virulent and a less virulent strain of the entomopathogenic fungus Beauveria bassiana, using restriction fragment length polymorphisms.

The genomic DNA of two strains of the entomopathogenic fungus Beauveria bassiana, strain GK2016, a "wild type" (virulent), and strain GK2051, a less virulent mutant to grasshoppers, was digested with 12 restriction endonucleases. Gel electrophoresis conditions were established to show restriction fragment length patterns visually in the digested DNA stained with ethidium bromide. The less virulent mutant was generated by ultraviolet illumination of conidiospores at a 95% lethal dose. Both strains of the fungi were identical in morphology as well as in 16 of 22 API-ZYM kit enzyme assays. Differences in levels of total enzyme activity were observed for esterase, esterase-lipase, beta-galactosidase, chitinase, and protease. A Neurospora crassa beta-tubulin gene (heterologous gene) and two homologous DNA probes (pJK16 and pJK18) hybridized to several specific DNA bands in B. bassiana strain GK2016 but not in strain GK2051. Strain GK2051 gave different restriction fragment length pattern when compared with its parent strain. Taken together, the data show restriction fragment length differences between the genomic DNA of the two strains, including the loss of some DNA sequences from the mutant strain, which may be involved in pathogenicity. Finally, B. bassiana GK2016 contains a beta-tubulin gene with at least partial homology to that of N. crassa.

Animals↗
PubMed1999

Diagnosis of a case of gastric anisakidosis by PCR-based restriction fragment length polymorphism analysis.

A set of genetic markers, based on PCR-RFLPs of three diagnostic restriction enzymes (Hhal, Hinfl and Taql), which proved to be suitable for the identification of the species of the genus Anisakis, was used for the first molecular identification of a larva obtained by endoscopy in a case of gastric anisakidosis, in a 51 year old woman from Southern Italy. The analysis of the restriction profiles obtained allowed the larva to be identified as Anisakis pegreffii, one of the three sibling species of the A. simplex complex. PCR-RFLP proved to be a cost-effective and reliable tool for the exact identification of Anisakis larvae recovered from infected humans.

Anisakiasis↗