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PubMed2002

A msp1alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale isolates.

Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease which can be protected by vaccination with the less pathogenic Anaplasma species, A. centrale. Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries. A molecular test specific for A. marginale would be ideal for the identification of Anaplasma species in wild ruminants, as possible reservoirs of anaplasmosis, and to differentiate between A. marginale from A. centrale. A PCR assay was designed to amplify the major surface protein 1alpha gene of the rickettsial bovine pathogen, A. marginale both as an inter- and intra-specific test. The test did not amplify A. centrale or A. ovis, and discriminated A. marginale by amplifying repeat regions within the msp1alpha gene which vary in number between many isolates. The nested A. marginale amplicons varied in size from 630 to 1190bp representing one to eight internal repeats. All 22 Australian isolates tested amplified a 630bp product (one repeat) in contrast to all 19 non-Australian isolates tested. Eight sequences from Australian isolates from different geographical regions confirmed the conserved nature of the Australian A. marginale msp1alpha genes. The Australian 'repeat unit' MSP1a deduced amino acid sequence has been designated as Australian type 1. The msp1alpha PCR method developed here enabled the amplification and comparison of A. marginale isolates originating from North and South America, Africa, Israel and Australia. The method is sensitive and specific for A. marginale. Although additional msp1alpha products were amplified from at least two Australian isolates, the results suggest limited introduction of A. marginale into Australia.

Amino Acid Sequence↗

PubMed1997

Mutations in the nonstructural protein 5A gene and response to interferon therapy in young patients with chronic hepatitis C virus 1b infection.

A region associated with sensitivity to interferon (IFN) has been identified previously in the nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) genotype 1b. A study was undertaken to determine whether the presence of mutations in the NS5A2209-2248 sequence could serve as a predictor of response to IFN therapy in children and adolescents with chronic HCV-1b infection. Sixteen children (M/F ratio = 11:5; mean age 11.7 years, range 5 to 19 years) with chronic HCV-1b infection who received IFN-alpha for 6 months (total dose: 8 MU/kg) were enrolled in this study. Twelve of the children (75%) had an underlying malignant disease. Pretreatment NS5A gene sequences were detected by reverse transcription-nested polymerase chain reaction (RT-nested PCR). PCR products were subjected to direct sequencing by the dideoxy chain termination method. The amino acid sequences of NS5A2209-2248 were compared with the published NS5A2209-2248 sequences of HCV-J. The NS5A2209-2248 sequences were detected in 10(63%) of the 16 children. Eight patients had the wild-type sequences, with no amino acid changes; and two patients had the intermediate type, with only one amino acid change. Four (25%) of the 16 patients responded completely to IFN therapy. Three of the four patients had the wild-type sequences, while none of the patients with the mutant type had a complete response. Serum HCV RNA levels in children with the wild type did not differ from those in patients with the mutant type. This study shows that there is no significant correlation between response to IFN and mutations in NS5A2209-2248. The amino acid sequences in NS5A2209-2248 in young patients with chronic HCV-1b infection appear to be conserved.

Adolescent↗

PubMed2003

[Detection of cell-free fetal DNA in the urine of pregnant women by nested polymerase chain reaction].

OBJECTIVE: To investigate the clinical feasibility of fetal sex determination by using nested polymerase chain reaction (PCR) to detect cell-free fetal DNA in the urine of pregnant women. METHODS: The urine specimens of 40 healthy predelivery women were centrifuged. The DNA in the specimens of supernatant was extracted by using of QIAamp Viral RNA Mini Kits. Nested PCR was used to specifically amplify the SRY gene, 300 bp in length, on Y chromosome in order to detect the fetal DNA from the women bearing male fetuses. One specimen of normal man's urine and one specimen of non-pregnant woman's urine were used as controls for each specimen from pregnant women. The results were confirmed by the sexes of neonates examined after birth. RESULTS: Amplified band of SRY was found in 6 specimens. Sex examination after delivery showed 21 male neonates and 19 female neonates. The positive rate of detection of SRY gene was 28.6% (6/21) and the false negative rate was 71.4%, and false positive rate was 0. Five out of the 6 cases with positive results showed positive 2 times and one out of the 6 cases only showed positive at the second time. CONCLUSION: There is cell-free fetal DNA in the urine of pregnant women. Nested PCR is useful in detection of fetal sex, however, still with a rather low positive rate.

DNA↗

PubMed2006

[Analysis of DEK-CAN fusion gene expression in acute myeloid leukemia patients with 6; 9 chromosome translocation].

This study was aimed to explore the relationship of 6; 9 chromosome translocation with DEK-CAN fusion gene expression in patients with acute myeloid leukemia (AML) and its clinical significance. Chromosome specimens were prepared by routine method after short-term culture of bone marrow cells; karyotype analysis was performed by R banding technique; the expression of fusion gene DEK-CAN was analyzed by RT-nested-PCR in mononuclear cells of bone marrow or peripheral blood of 4 AML patients, for 3 patients received allo-BMT out of 4 patients the dynamic follow-up was performed. The results indicated that t (6; 9) (p23; q34) was confirmed by chromosome karyotype analysis in the four AML patients. The DEK-CAN fusion gene was found during in all four de novo, relapsed and CR patients (100%). And the expression of DEK-CAN fusion gene enhanced apparently in de novo and relapsed patients, and weakened in CR patient. DEK-CAN mRNA was found in the three patients during 1-24 months after allo-BMT. Clinical data showed 2 patients relapsed and died after CR for 1-24 months; the other two patients received allo-BMT got CR and still survive. It is concluded that DEK-CAN fusion gene is the molecular basis in pathogenesis of AML. The detection of DEK-CAN fusion gene is significant for diagnosis of AML, evaluation of curative effect, and predication of prognosis.

Acute Disease↗

PubMed1997

Primary structure and variability of partial nonstructural gene 5 region of hepatitis G virus.

OBJECTIVE: To sequence partial nonstructural 5 (NS5) gene of hepatitis G virus (HGV) in the sera of Chinese patients with chronic non-A-E hepatitis or hepatitis B virus (HBV) infection. METHODS: Total nucleic acid was extracted from the sera of 35 patients with chronic non-A-E hepatitis and 20 patients with HBV infection, then it was subjected to reverse transcriptase-nested-polymerase chain reaction (RT-nested-PCR) with primers derived from the putative nonstructural gene 5 (NS5) region of HGV. The positive PCR products (994 bp in length) were directly sequenced using the Sang's method after purification. RESULTS: HGV RNA was detected in 1 of the 35 patients (2.9%) with chronic non-A-E hepatitis and 5 of the 20 patients (25%) with HBV infection. All the 6 positive samples were sequenced. These 6 Chinese isolates exhibited 87.21% to 93.92% nucleotide sequence identities with 3 reported isolates (GBV-C, PNF2161, R10292) over the 994 nucleotides examined, while the nucleotide homologies among the 6 isolates were 90.09% to 93.82%. And 93.61% to 98.72% homologies were found at the deduced amino acid level in comparing the 6 isolates with 3 reported ones, while 93.61% to 98.40% amino acid identities were found among the 6 isolates. In the region sequenced, there existed 16 conserved proline residues and 8 conserved cysteine residues. CONCLUSIONS: Six isolates of HGV were obtained from different Chinese patients in this study. The partial nucleotide sequences, as well as amino acid sequences, of the putative NS5 region of HGV in Chinese patients are relatively conserved. The overall degree of nucleotide conservation (90.09% to 93.82%) among Chinese isolates may make this region of the genome suitable for the development of diagnosis reagents.

Amino Acid Sequence↗

PubMed1999

[The relationship between the persistence of the BCR/ABL gene and relapse in adult patients with acute lymphoblastic leukemia].

BACKGROUND: The Philadelphia chromosome (Ph') is originated by the t(9;22) which determines the rearrangement BCR/ABL. This rearrangement has been associated with an unfavourable prognosis in patients diagnosed with adult acute lymphoblastic leukaemia (ALL). PATIENTS AND METHODS: The BCR/ABL gene (p210 and p190) was prospectively studied by nested RT-PCR in 17 adult patients diagnosed with ALL BCR/ABL-positive cases were monitored by RT-PCR and cytogenetic techniques over the treatment period (LAL-93 AR protocol). RESULTS: BCR/ABL mRNA was detected in 8 out the 17 patients studied (47%). The Ph' chromosome was detected in 4 cases. Follow-up was completed in 6 out of the 8 BCR/ABL positive cases. PCR only became negative in one patient. The 5 patients with persistently positive BCR/ABL relapsed, whereas the case which became negative was still in complete remission after 24 months follow-up. In 3 out of the 4 Ph' positive patients, the karyotype was normal after induction therapy. CONCLUSIONS: This study clearly demonstrates the usefulness of molecular analysis in the diagnosis and follow-up of ALL compared with conventional cytogenetic techniques. The importance of molecular analysis to assess the efficacy of the treatment used has been emphasized and the poor evolution of BCR/ABL-positive patients has been confirmed.

Adolescent↗

PubMed2000

Analysis of 16S rRNA and 51-kilodalton antigen gene and transmission in mice of Ehrlichia risticii in virgulate trematodes from Elimia livescens snails in Ohio.

Operculate snails (the family Pleuroceridae: Elimia livescens) were collected between June and October 1998 from a river in central Ohio where repeated cases of Potomac horse fever (PHF) have occurred. Of collected snails, consistently 50 to 80% carried a combination of cercariae and sporocysts of digenetic virgulate trematodes. The trematodes obtained from each snail were pooled and tested for Ehrlichia risticii, the agent of PHF, by nested PCR using primers specific to the 16S rRNA gene. Out of a total of 209 trematode pools, 50 pools were found to be positive by PCR. The DNA sequence of the 16S rRNA gene identified in one trematode pool was identical to that of the type strain of E. risticii, and the sequence of the gene identified in another pool differed from that of the type strain by 1 nucleotide. Comparison of the deduced amino acid sequence of the partial 51-kDa antigen gene from various sources revealed that Maryland, Ohio (except Ohio 081), and Kentucky strains are in a cluster distinct from the sequences obtained from sources in California and Oregon. Ohio 081 was shown previously by antigenic composition analysis to be distinct from other groups. However, all sequences examined were not segregated according to their sources: horse blood or infected trematodes. E. risticii was found to be transmittable from trematodes to mice and was subsequently passaged from infected mice to additional mice, as determined by PCR analysis. Our findings suggest the evolution of E. risticii in the natural reservoir in separate geographic regions and persistent infection of trematode populations with E. risticii during summer and early fall. The study also suggests that the mouse can be used to isolate E. risticii from the infected trematode.

Amino Acid Sequence↗

PubMed1996

An endo-beta-1,4-glucanase gene (celA) from the rumen anaerobe Ruminococcus albus 8: cloning, sequencing, and transcriptional analysis.

A genomic library of Ruminococcus albus 8 DNA was constructed in Escherichia coli using bacteriophage lambda ZapII. This library was screened for cellulase components and several Ostazin brilliant red/carboxymethyl cellulose positive clones were isolated. All of these clones contained a common 3.4-kb insert, which was recovered as a plasmid by helper phage excision. The carboxymethyl cellulase coding region was localized to a 1.4-kb region of DNA by nested deletions, and a clone containing the entire celA gene was sequenced. Analysis of the sequence revealed a 1231-bp open reading frame, coding for a protein of 411 amino acids with a predicted molecular weight of 45 747. This protein, designated CelA, showed extensive homology with family 5 endoglucanases by both primary amino acid sequence alignment and hydrophobic cluster analysis. Cell-free extracts of E. coli containing the celA clone demonstrated activity against carboxymethyl cellulose and acid swollen cellulose but not against any of the p-nitrophenol glycosides tested, indicating an endo-beta-1,4-glucanase type of activity. In vitro transcription-translation experiments showed that three proteins of 48,000, 44,000, and 23,000 molecular weight were produced by clones containing the celA gene. Northern analysis of RNA extracted from R. albus 8 grown on cellulose indicated a celA transcript of approximately 2700 bases, whereas when R. albus 8 was grown on cellobiose, celA transcripts of approximately 3000 and 600 bases were detected. Primer extension analysis of these RNAs revealed different transcription initiation sites for the celA gene when cells were grown with cellulose or cellobiose as the carbon source. These two sites differed by 370 bases in distance. A model, based on transcription and sequence data, is proposed for celA regulation.

Amino Acid Sequence↗

PubMed1997

Dystrophin gene transcripts skipping the mdx mutation.

The mdx mouse, an animal model used to study Duchenne muscular dystrophy, has a nonsense mutation in exon 23 of the dystrophin gene which should result in a truncated protein that cannot be correctly localized at the sarcolemma of the muscle fibers. Immunohistochemical staining with antidystrophin antibodies has shown that while most of the muscle tissue is dystrophin-negative, a small percentage of muscle fibers is clearly dystrophin-positive and has somehow bypassed the primary nonsense mutation. A sensitive nested polymerase chain reaction-based examination of dystrophin gene transcripts around the mdx mutation has revealed several alternatively processed transcripts. Four mRNA species skipped the mutation in exon 23, were in-frame, and could be translated into a shorter but still functional dystrophin protein. Specific tests for these transcripts demonstrated these were also present in normal mouse muscle tissue.

Animals↗

PubMed2002

Two distinct types of hepatitis B virus core promoter variants in Yemeni blood donors.

Genetic variations in the basic core promoter (BCP) region of hepatitis B virus (HBV) occur during the natural history of chronic HBV infection. This study investigates the presence of basic core promoter variations in 28 asymptomatic Yemeni blood donors, correlating variations with HBeAg phenotype and viral load. The core promoter/precore and surface gene region of HBV DNA were amplified using nested PCRs and the PCR products were sequenced either directly or after cloning. HBeAg and viral load were measured when HBV DNA was detectable. Sequencing of 18 surface PCR products indicated that all were of genotype D. Two distinct types of variants were identified in the basic core promoter: substitution only (N = 14) and major deletion (N = 9). The commonest substitutions were located at nucleotide positions 1753, 1762, and 1764; 10/14 (71.4%) were associated with the precore 1896A substitution resulting in the premature stop of the precore reading frame and 6/14 (42.9%) had viral loads above 400 copies/ml. Two forms of deletion variants were found: 8 bp deletion (1763-1770) (N = 2) and a novel 12 (1746-1757) + 8 bp (1763-1770) deletion (N = 7). The deletion sequences were never associated with the precore 1896A substitution and all had viral load below 400 copies/ml with negative HBeAg phenotype. The polymorphism 1773C was found in 9/14 (64.3%) substitution sequences whereas all deletion sequences had 1773T. Two donors had mixed sequences of basic core promoter substitution and major deletions (both 8 bp and 12 + 8 bp). While the deletion variants in these two donors were similar to others found in isolation, the substitutions were of a different pattern. Further studies are required to understand the selection process behind these variants.

Adolescent↗

PubMed2001

Detection of horses infected naturally with equine infectious anemia virus by nested polymerase chain reaction.

A nested polymerase chain reaction (PCR) amplifying a region of the gag gene of equine infectious anemia virus (EIAV) was developed for the rapid and direct detection of proviral DNA from the peripheral blood of naturally infected horses and was compared with the Coggins test. DNA prepared from white blood cells of 122 field horses from 15 stables with reported cases of EIAV and one seronegative stable were analysed. Amplifications of expected size fragments were obtained by nested PCR for 88 horses using two different sets of primers targeting the gag region. The specificity of the amplified products was confirmed by hybridization using a digoxigenin-labeled probe. Gag-nested PCR-restriction fragment length polymorphism analysis distinguished two different subtypes of gag gene, A and B. Subtype A was found to be the most prevalent among the infected horses that were tested. The PCR-gag amplified sequence of subtype A shared 84.6% nucleotide and 93% deduced amino acid sequence identities with the prototype Wyoming strain whereas subtype B sequence was almost 100% identical to the prototype. Sequence analysis of gag subtype A suggests the presence of a novel EIAV variant among infected horses in Canada. The nested PCR assay developed in the present study detected more EIAV positive animals and was found as specific as the agar gel immunodiffusion (Coggins) assay and offers great potential a diagnostic test for the detection of EIAV infections in field horses.

Animals↗

PubMedDate not supplied

Prenatal diagnosis of achondroplasia using the nested polymerase chain reaction with modified primer sets.

Achondroplasia (ACH) is the most frequent form of short-limb dwarfism. Recently, the gene mutation responsible for ACH has been identified in the transmembrane domain of the fibroblast growth factor receptor 3 gene. The cause of ACH is a point mutation at nucleotide 1138 of the cDNA, resulting in the substitution of an arginine residue for a glycine. For the purpose of prenatal diagnosis of ACH, we have used the nested polymerase chain reaction to ensure the gene amplification. Although the normal allele has no restriction site around the causative sequence, we have devised an unique method to incorporate a restriction site of SplI into the normal allele only using modified primer sets. We report here the use of this polymerase chain reaction methodology which can ensure the gene amplification and omit the complicated steps of sequencing for prenatal diagnosis.

Alleles↗

PubMed1992

Nested polymerase chain reaction on cellular DNA in plasma: a rapid method to investigate the collagen type I A2 MspI polymorphic restriction site in alcoholic patients.

We investigated the MspI restriction site of the human collagen type I A2 (COL1A2) gene in alcoholic subjects with and without liver disease, using the nested polymerase chain reaction to analyse trace cellular DNA in plasma samples. This procedure is rapid, it requires only 200 microliters plasma, and the results correlate perfectly with those of Southern blotting. Alcoholic subjects with acute alcoholic hepatitis had a significantly lower frequency of the minor allele than healthy controls or alcoholics with minimal liver abnormalities, i.e. steatosis. Our results reinforce the hypothesis of a genetic predisposition of certain alcoholics to liver damage.

Alcoholism↗

PubMed1996

Detection of HIV-1 DNA in needle/syringes, paraphernalia, and washes from shooting galleries in Miami: a preliminary laboratory report.

Shared use of injection equipment (needle/syringes), registering, booting, and backloading are practices among injection drug users (IDUs) that increase the risk for transmission of human immunodeficiency virus type 1 (HIV-1). The sharing of injection paraphernalia (including cookers and cottons) and washwater for rinsing used needle/syringes and dissolving drugs could be potential sources for secondary transmission of HIV-1. Laboratory rinses were made from needle/syringes, cottons, and cookers obtained from shooting galleries, and washwaters were obtained from shooting galleries in Miami. Three rinses were analyzed and antibodies to HIV-1 proteins were detected by using Western blot and HIV-1 DNA was detected by using nested polymerase chain reaction (PCR) specific for the gag and envelope genes of HIV-1. Antibodies to HIV-1 proteins were detected in 12 (52%) of 23 rinses from visibly contaminated needle/syringes, in three (18%) of 17 rinses from cottons, in three (14%) of 21 rinses from cookers, and in one (6%) of 17 washwaters. No antibodies were detected in laboratory rinses from visibly clean needles. Using nested PCR followed by Southern blot confirmation of the amplified targets, HIV-1 gag gene DNA was detected in 16 (84%) of 19 and envelope gene DNA in 17 (85%) of 20 laboratory rinses from visibly contaminated needle/syringes. We detected gag and envelope gene DNA, respectively, in three (27%) and four (36%) of 11 cottons, in six (46%) and seven (54%) of 13 cookers, and in five (38%) of 13 and in 10 (67%) of 15 washwaters from shooting galleries. No HIV-1 DNA was detected in laboratory rinses from visibly clean needles. These results indicate that HIV-1 might be present in contaminated cottons, cookers, and washwaters as well as in contaminated needle/syringes at shooting galleries. Reduction of risks of exposure to HIV-1 among IDUs may require modification of behaviors that are ancillary to the act of injection, such as the use of common cookers, cottons, and washwater.

Blotting, Southern↗

PubMed1991

Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes.

Using 10 overlapping nested sets of primers and using peripheral blood lymphocyte (PBL) total RNA as template, we have developed a system, based on PCR, which allows the rapid production of double-stranded cDNA corresponding to the entire coding sequence of the dystrophin gene. The product can be visualized on native minigels by ethidium staining and directly sequenced after gel purification. We have used this system to analyze the structures of PBL dystrophin mRNA in 26 Duchenne, Becker, or intermediate muscular dystrophy patients who have gross rearrangements of the dystrophin gene. In each case, the effect that the genomic rearrangement has on the structure of the transcript--and, by inference, on the dystrophin protein--has been determined, and the results confirm the frameshift hypothesis. The study also identifies a series of alternatively spliced transcripts which are specific to the rearranged genotypes and which seem therefore to arise following the alteration in the context of the splice signal. The system has been used for unambiguous identification of carrier females. Furthermore, the rapid production of microgram quantities of dystrophin cDNA from a readily accessible tissue makes point-mutation screening a practical proposition.

Base Sequence↗

PubMed1997

Quantification of hexokinase mRNA in mouse blastocysts by competitive reverse transcriptase polymerase chain reaction.

Hexokinase (HX), the enzyme that catalyses the initial reaction in glycolysis, is an important enzyme in glucose metabolism during human and mouse embryonic development. In our previous investigations of the genetic activities of HX, we observed an increased incidence of HX gene expression in blastocysts in comparison with morulae, and variability in the incidence of HX gene expression between embryos at the same developmental stages. These observations prompted us to quantify HX mRNA in mouse blastocysts to define the biological significance of the variable gene transcription. We modified our qualitative reverse transcription-nested polymerase chain reaction (RT-nPCR) assay for HX mRNA in single or groups of embryos to quantify HX mRNA by competitive RT-nPCR. HX mRNA was quantified in cohorts of mouse blastocysts cultured in glucose/phosphate-containing human tubal fluid (HTF) media. These blastocysts expressed HX in minute amounts, averaging 1.95 x 10(-18) g of mRNA. This is the first attempt at quantification of single gene mRNA in preimplantation embryos. Further investigations using similar techniques will enable comparative analyses between embryos to be performed to determine the correlation between specific levels of HX mRNA transcripts in individual embryos and embryonic viability and competence for further development and implantation.

Animals↗

PubMed2002

[Detection of multiple loci in single cell by primer extension preamplification and nest PCR]

OBJECTIVE: To investigate the feasibility of multiple loci detection in single cell by primer extension preamplification (PEP) followed by nest PCR. METHODS: Using PEP, the whole genomic DNA in single lymphocyte or single blastomere was amplified. In addition, CD17, nt-28 and linked ATTTT repeat for beta-thalassemia, F508 and linked GATT repeat for cystic fibrosis, DMD exon 17 and 48 for Duchenne muscular dystrophy, short tandem repeats of D18S51, D21S11 and D21S1411, and sex-determination gene SRY of the Y chromosome were all detected using nest-PCR from a small aliquot of the PEP reaction. RESULTS: The rate of successful single lymphocyte amplification was 89.5%(false positive 0.48%false negative 2.5%). The rate of successful single blastomere amplification was 85.56%(false positive 3%). CONCLUSION: The PEP technique followed by nest PCR analysis of single cell is very useful for simultaneous detection of multiple gene loci. It may be applicable for preimplantation genetic diagnosis.

Journal Article↗

PubMed2005

Behavioral risk exposure and host genetics of susceptibility to HIV-1 infection.

BACKGROUND: Some individuals are readily infected with low human immunodeficiency virus type 1 (HIV-1) exposure, whereas others appear less susceptible, suggesting that host genetics plays a role in the viral entry pathway. The matched case-control study design with measured risk exposures provides an avenue for discovering genes involved in susceptibility to infection. METHODS: We conducted a nested case-control study of African Americans (266 HIV-1 seroconverter cases and 532 seronegative controls from the AIDS Link to Intravenous Experience cohort), to examine the association between 50 single-nucleotide polymorphisms (SNPs) in 9 candidate genes (CCR5, CCR2, RANTES, MIP1A, MCP2, IL10, IFNG, MCSF, and IL2) and susceptibility to HIV-1 infection. To account for differential exposure propensities, risk behavior self-reported during semiannual visits was used to estimate a standardized cumulative risk exposure (SCRE). Individual SNPs were evaluated using conditional logistic-regression models, and the inferred haplotypes were assessed in the haplotype trend regression analyses after adjusting for age and SCRE. RESULTS: Four SNPs (CCR2-V64I, CCR5-2459, MIP1A+954, and IL2+3896) and specific haplotypes in the IL2 and CCR2/CCR5 regions were significantly associated with HIV-1 infection susceptibility in different genetic models. CONCLUSIONS: Our results suggest that genetic variants in associated host genes may play an important role in susceptibility to HIV-1 infection.

Adult↗