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PubMed1995

[The 69th annual meeting president lecture. Application of molecular biology for rapid detection of mycobacteria].

The application of molecular biology techniques for the rapid detection of mycobacteria in clinical specimens was evaluated. The DNA probe method was found to be accurate and rapid for identification of mycobacteria, but its low sensitivity needs improving. The nested polymerase chain reaction (PCR) targeting Pab gene was specific and sensitive enough for rapid detection of Mycobacterium tuberculosis in clinical specimens. The overall sensitivity and specificity of this method were excellent, 97% and 92%, respectively. Additionally, a novel method for the rapid detection of mycobacteria other than M. tuberculosis was developed using a combined method of PCR and DNA probe. The gene encoding 16S ribosomal RNA in mycobacteria was first amplified by PCR, and then PCR products were identified using an acridinium-ester labeled DNA probe. This method was as useful as that of the PCR for M. tuberculosis described above. The DNA probe method was also applied for drug sensitivity testing, which needs three to four weeks by conventional methods. The quantity of ribosomal RNA in mycobacteria cultured with or without the addition of drugs was compared. Three days after the start of culture, the strains sensitive for isoniazid or rifampicin showed remarkable decreases of ribosomal RNA, compared with the drug-free samples, while the resistant strains showed no difference. Another method for determining the drug resistance of mycobacteria consists of detection of the gene which is related to the resistance. The deletion of catalase-peroxidase gene suggested a gene related to isoniazid resistance was observed in 15% of isoniazid-resistant strains.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗

PubMed1996

Molecular diagnosis of mantle cell lymphoma in paraffin-embedded tissue.

Mantle cell lymphoma (formerly known as intermediate lymphocytic lymphoma, diffuse centrocytic lymphoma, or diffuse small cleaved lymphoma) is one of the small cell non-Hodgkin lymphoma entities that is clinically more aggressive than small lymphocytic lymphoma, and needs to be separated from it. Mantle cell lymphoma is strongly associated with the t(11;14) chromosomal translocation that rearranges the bcl-1 oncogene (PRAD-1 gene) and immunoglobulin heavy chain gene. In this study, we developed a nested polymerase chain reaction system to evaluate the t(11;14) translocation. The study material consisted of 10 mantle cell lymphomas fulfilling the criteria suggested by other authors (P. M. Banks et al. Surg Pathol 16:637, 1992). A novel nested polymerase chain reaction system was used to evaluate the bcl-1 breaks in the major translocation cluster using two successive polymerase chain reaction amplifications. This reaction yielded a background-free single product of the size of 200 to 300 base pairs in four of 10 mantle cell lymphomas. The identity of the product from the nested polymerase chain reaction was confirmed by Southern blotting followed by hybridization with a specific probe. The amplification products were also evaluated by Sst-I, Alu-I, Dde-I, and Ita-I restriction enzymes and showed different patterns of digestion reflecting individual differences between the MTC/ IgH junctions. A selection of other low-grade lymphomas, including lymphocytic, follicular, and mucosa-associated lymphoid tissue lymphoma, and hairy cell leukemia and 29 hyperplastic lymph nodes were negative. This nested polymerase chain reaction system for the t(11;14) translocation involving major translocation cluster offers a convenient specific identification for mantle cell lymphoma. However, this test has a limited diagnostic power because only about half of the mantle cell lymphomas show the bcl-1 breaks in the major translocation cluster. The test performs well in formaldehyde-fixed and paraffin-embedded material, allowing the study of large numbers of retrospective cases of mantle cell lymphomas.

Aged↗

PubMed1998

Detection of bcr/abl mRNA in a case of chronic myelogenous leukemia in long-term remission: CML or sensitivity of detection?

Chronic myelogenous leukemia (CML) is a myelo-proliferative disorder which, after a chronic phase which lasts an average of 3 years, evolves into an acute disease which is resistant to chemotherapy. Nevertheless, a few studies have reported cases in which partial or complete hematologic, cytogenetic and/or molecular remission of the disease were observed either spontaneously or after non intensive chemotherapy, with or without medullar aplasia. Some of these patients later relapsed into a blast crisis. We report a case of CML with clinical and hematologic remission for 19 years after two cycles of busulphan not causing medullar aplasia, negative for the BCR/ABL gene by Southern blot but with the gene's mRNA detectable by hot start nested RT-PCR.

Adult↗

PubMed1998

Detection of HIV-1 infection in dried blood spots from a 12-year-old ABO bedside test card.

BACKGROUND AND OBJECTIVES: We tested dried blood from an ABO bedside test card which had been stored at room temperature for 12 years, to prove that a patient with HIV-1 infection had been infected by blood transfusion. MATERIALS AND METHODS: Immunoblots for HIV-1 antibodies and threefold PCRs with half-nested primers for the HIV-1 integrase gene were done with eluates from the dried blood spots. RESULTS: HIV-1 antibodies and HIV-1 DNA could be detected in the sample from one unit of blood, but not from the two other units or from the recipient before transfusion. CONCLUSION: Further studies should be done on the validity of stored dried blood as an alternative to the storage of frozen donor serum for several years for 'look-back' studies.

ABO Blood-Group System↗

PubMed2004

Detection and identification by real time PCR/RFLP analyses of Cryptosporidium species from human faeces.

AIMS: To detect a wide range of Cryptosporidium species from human faeces by analysis of the Cryptosporidium oocyst wall protein gene by PCR. METHODS AND RESULTS: The nested-assay comprised an initial amplification using a conventional thermocycler followed by real time PCR using a LightCycler with SYBR Green I for the characterization of the amplicons. The technique uses four sets of primers composed of five to six oligonucleotides with one to six base differences corresponding to the inter-species sequence differences of the gene fragment. Restriction fragment length polymorphism analysis identified Cryptosporidium hominis and C. parvum. The assay was evaluated using DNA extracted from purified material and faecal specimens containing a range of potential pathogens (including Cryptosporidium). The assay was specific, sensitive, reproducible and rapid. CONCLUSIONS: This unique technique enables the rapid detection of a range of polymorphic COWP gene sequences directly from faeces using real time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a novel approach to identification of Cryptosporidium species and the identification of C. hominis and C. parvum. The technique may be especially useful for the analysis of environmental samples which are likely to contain heterogeneous mixtures of Cryptosporidium species.

Animals↗

PubMed1996

Population genetics with RAPD-PCR markers: the breeding structure of Aedes aegypti in Puerto Rico.

RAPD-PCR polymorphisms at 57 presumptive loci were used to examine the breeding structure of the mosquito Aedes aegypti in Puerto Rico. Mosquitoes were sampled from 16 locations in six cities and samples were located in a nested spatial design to examine local patterns of gene flow. Allele frequencies were estimated assuming (1) that genomic regions amplified by RAPD-PCR segregate as dominant alleles, (2) that genotypes at RAPD loci are in Hardy-Weinberg proportions, (3) identity in state (iis) among dominant amplified alleles and (4) iis among null alleles. The average genic heterozygosity was 0.354, more than twice the level detected in earlier allozyme surveys. Nested analysis of variance indicated extensive genetic differentiation among locations within cities. Effective migration rates (Nm) among cities were estimated from FST assuming an island model of migration. Estimates of Nm ranged from 9.7 to 12.2 indicating a high dispersal rate. The large number of polymorphisms revealed by RAPD-PCR allowed the distribution of FST and linkage disequilibrium to be examined among loci and demonstrated that small samples inflate FST and linkage disequilibrium. No linkage disequilibrium maintained through epistasis was detected among alleles at the 57 loci.

Aedes↗

PubMed2005

AT1 receptor A1166C and AT2 receptor -1332A/G gene polymorphisms: efficient genotyping by single-tube PCR.

Angiotensin II type 1 receptor (AT1) and angiotensin II type 2 receptor (AT2) genes have been investigated in recent years as potential etiologic candidates for cardiovascular and renal diseases. The pathogenic implications of AT1 A1166C and AT2 A-1332G gene polymorphisms have been shown. Here we describe a rapid and reliable method for detecting both AT1 and AT2 gene polymorphisms by a single-tube PCR, to reduce analysis time and simplify the genotyping procedure. In contrast to previously described methods, our method does not require hybridization, primer extension, or nested PCR for genotyping. In most previous studies concerning gene polymorphisms of RAS, both AT1 and AT2 receptor gene polymorphisms were investigated. The advantage of our method is that it makes it possible to detect both of these polymorphisms in a duplex PCR. The procedure described is convenient for routine laboratory use with manual sample processing, and offers the potential for further automation as well. Its simplicity makes it practical for large-scale screening of individuals and families at risk for cardiovascular or renal diseases.

Cardiovascular Diseases↗

PubMed1996

Genital chlamydial infection among women in Nicaragua: validity of direct fluorescent antibody testing, prevalence, risk factors and clinical manifestations.

OBJECTIVE: To validate the performance of a direct fluorescence antibody (DFA) test and to determine the prevalence, risk factors and clinical manifestations of cervical chlamydia infection in different groups of women in Nicaragua. STUDY POPULATION: 926 women, 863 routine clinic attenders (mean age 27 years) and 63 sex workers (mean age 25 years) attending health centres in León, Corinto, Matagalpa and Bluefields. METHODS: Cervical specimens were examined using the Syva MicroTrak test system with a cut-off of 10 or more elementary bodies (EBs). The DFA results were validated by a one-step polymerase chain reaction (PCR) assay. Discordant results were further examined in nested PCR assays directed at two different target genes. An interviewer-administered questionnaire and a standard gynaecological examination were completed. RESULTS: Sensitivity of DFA was 80.1%, specificity 98.3%, and positive and negative predictive values 62.5% and 99.3%, respectively. Values were lower in locations where samples thawed because of electricity breaks and higher among sex workers. The majority of discordant results was confirmed as positive in nested PCR assays. Prevalence of cervical chlamydia infection based on positivity in DFA and/or PCR ranged from 2% among routine clinic attenders aged 35 years or older, to 8% among adolescent clinic attenders, and to 14% among sex workers. Among routine clinic attenders, young age (odds ratio [OR] 3.6, 95% confidence intervals [95% CI] 1.4-8.9 for women aged 15-19 years as compared with 1 in women 25 years of age or older) and use of oral contraceptives (OR 4.0, 95% CI 1.7-9.6) were the only statistically significant risk factors identified in multivariate logistic regression analysis. Presence of mucopurulent cervical discharge (OR 5.9, 95% CI 3.0-11.5) and presence of ectropion (OR 2.6, 95% CI 1.1-6.5) were the clinical signs independently associated with infection. CONCLUSIONS: Our results indicate that the DFA test was sensitive and specific while the performance of the PCR assay depends on adequate storage of samples. Genital C trachomatis infection is a common health problem among women in Nicaragua. The wide implementation of syndromic STD management algorithms together with health education programmes aimed at young people is the most promising approach to control STD in Nicaragua.

Adolescent↗

PubMed1995

Prenatal sex determination in Thai pregnant subjects by nested-PCR amplification of fetal Y chromosome-specific sequence.

Detection of a Y-specific sequence in either amniotic cells or the maternal circulation has clinical importance for prenatal sex diagnosis, especially in mothers with severe X-linked disorders. Deoxyribonucleic acid sequences of human ZFY (zinc-finger-Y) gene, a Y-chromosome-specific gene and candidate for the testis-determining factor, were amplified by in vitro nested PCR technique, in amniotic cells or peripheral blood of Thai pregnant subjects carrying male fetuses. This technique permits detection of ZFY gene DNA sequences in as few as a single male cell among 100,000 female cells. The subjects in this study were a group of Thai pregnant women who had been given prenatal fetal sex diagnosis by the cytogenetic method. The results herein demonstrated the usefulness of PCR detection of Y-specific sequences in amniotic cells. The ZFY gene detection method for prenatal sex prediction by nested PCR has been developed experimentally. However, further investigation is necessary to increase reliability of clinical application. With properly designed guidelines, the established nested-PCR technique may be an alternative for the determination of the fetal sex for those pregnancies at risk of X-linked genetic disorders.

Amniocentesis↗

PubMed1982

Nest-building behavior in two cerebellar mutant mice: staggerer and weaver.

Nest-building behavior was studied in two separate (different genes) but phenotypically similar autosomal recessive neurological (cerebellar) mutants, staggerer and weaver, as well as their wild-type siblings. None of the test animals or their parents had previous experience with the nesting materials used in these experiments. All of the mice, with the exception of the staggerer mutant, exhibited the nest-building behavior. This observation, coupled with other phenotypes expressed in this mutant, suggest the mutation may more generally affect neuroendocrine development and/or regulation rather than be restricted to cerebellar development.

Animals↗

PubMed1994

Survey on the distribution of the gene 4 alleles of human rotaviruses by polymerase chain reaction.

The presence of six gene 4 alleles (or VP4 genotypes) in human rotaviruses has been recognized. Using 16 representative cultivable human rotavirus strains, we confirmed the specificity of VP4 genotyping by polymerase chain reaction (PCR) using the nested oligonucleotides specific to each of the four representative gene 4 alleles. Using the PCR, we surveyed the gene 4 alleles of 199 human rotaviruses in stools collected in Japan and Thailand. Strains with the gene 4 allele, corresponding to P1A serotype, were shown to be the most prevalent, but two strains with P2 gene 4 allele and one strain with P3 gene 4 allele were detected in Thailand and in Japan, respectively.

Alleles↗

PubMed2002

Baculoviruses exhibit restricted cell type specificity in rat brain: a comparison of baculovirus- and adenovirus-mediated intracerebral gene transfer in vivo.

Baculoviruses have recently been shown to be effective gene transfer vectors in mammalian cells. However, very little information is available about their target cell tropism in the central nervous system. We studied transduction efficiency, tropism and biodistribution of baculoviruses after local delivery to rat brain and compared their properties to adenoviruses. It was found that baculoviruses specifically transduced cuboid epithelium of the choroid plexus in ventricles and that the transduction efficiency was as high as 76+/-14%, whereas adenoviruses showed preference to corpus callosum glial cells and ventricular ependymal lining. Only a modest microglia response was seen after the baculovirus transduction whereas the adenovirus gene transfer led to a strong microglia response. Sensitive nested RT-PCR revealed transgene expression in the hindbrain and in ectopic organs including spleen, heart and lung, which indicates that some escape of both vectors occurs to ectopic organs after local gene transfer to the brain. We conclude that both baculovirus and adenovirus vectors can be used for local intracerebral gene therapy. The knowledge of the cell type specificity of the vectors may offer a possibility to achieve targeted gene delivery to distinct brain areas. Baculoviruses seem to be especially useful for the targeting of choroid plexus cells.

Adenoviridae↗

PubMed1996

Detection of human T-cell lymphotrophic virus type I in archival tissue specimens.

OBJECTIVE AND DESIGN: To develop a method for the detection of human T-cell lymphotropic virus type I (HTLV-I) in archival biopsy specimens. A polymerase chain reaction-based gene amplification method was developed to detect HTLV-I proviral DNA in paraffin-embedded specimens. The specificity of the polymerase chain reaction products was controlled by Southern blot analysis using a nested oligonucleotide probe and by nucleotide sequencing. The nucleophosmin gene and the T-cell receptor-gamma gene were used as controls for the integrity and adequacy of total DNA and T-cell DNA, respectively. This study was conducted with patients referred to an academic medical center. Biopsy specimens were obtained from lesional skin or lymph node from Japanese patients with HTLV-I seropositive adult T-cell leukemia/lymphoma. The main outcome measure was the ability to detect HTLV-I pX region proviral DNA. RESULTS: Comparative analysis of DNA extracted from fresh samples of the HTLV-I infected MT4T-cell line demonstrated that formalin fixation and paraffin embedding resulted in a 100-fold reduction in sensitivity of the assay. Nevertheless, HTLV-I pX sequences were still readily detectable in paraffin-embedded samples of MT4 T cells and adult T-cell leukemia/lymphoma specimens. Both formalin and B5 fixation were suitable for the assay that was 100% specific for HTLV-I-infected tissues. CONCLUSIONS: The use of this method should greatly facilitate investigation of the role of HTLV-I in human diseases by allowing analysis of a wide variety of archival tissue specimens. In addition, the controls designed for the current study can be used in a variety of other polymerase chain reaction-based studies of T cells to ensure against false-negative results caused by DNA degradation or inadequate T-cell density.

Adult↗

PubMed2002

T cell receptor repertoire of T cells in the kidneys of patients with lupus nephritis.

OBJECTIVE: To determine the role of T cells in the pathogenesis of lupus nephritis (LN). METHODS: Renal biopsy specimens from 12 patients with systemic lupus erythematosus were used for the experiments. We analyzed T cell receptor (TCR) Vbeta1-20 family genes on intrarenal T cells and on peripheral blood lymphocytes (PBLs) by nested reverse transcriptase-polymerase chain reaction (PCR) and Southern blot analysis. Nucleotide sequence was determined in the third complementarity-determining region of the TCR Vbeta gene in expanded T cells. Messenger RNA (mRNA) expression levels of Th1 and Th2 cytokines on infiltrating T cells were measured by nested PCR. RESULTS: The repertoire of TCR Vbeta in intrarenal T cells was relatively restricted compared with that in PBLs. The TCR Vbeta8 and TCR Vbeta20 genes were preferentially expressed in 6 of 12 patients (50%) and the TCR Vbeta9 and TCR Vbeta14 genes were expressed in 5 of 12 patients (42%). Junctional sequences of complementary DNA encoding the TCR Vbeta8 and TCR Vbeta20 genes in intrarenal T cells showed oligoclonal expansion, indicating antigen-driven stimulation. Interleukin-4 (IL-4) and IL-10 mRNA were highly expressed on intrarenal T cells, while interferon-gamma mRNA was not detected. CONCLUSION: Our findings suggest that T cells infiltrating the kidneys of patients with LN may recognize restricted epitopes on antigens and function as Th2-type T cells.

Adolescent↗

PubMed2002

A novel molecular method for HIV-1 proviral DNA detection: non-radioactively--reversed probe hybridization and nested PCR.

A novel molecular method for HIV-1 proviral DNA detection comprising two main techniques: nested PCR, amplifying a target sequence of the ENV-gene of HIV-1, and nonradioactively-reversed probe hybridization for the detection of the amplified target sequence. The dual amplification of inserted HIV-1 proviral DNA in each DNA sample to be tested was performed by nested PCR in two steps: firstly with two outer primers covering the target sequence of the ENV-gene of HIV-1; secondly with two 5'-biotinylated primers specific to the target sequence. The biotinylated PCR product could be visualized as a single band of 141bps in length on agarose gel stained with ethidium bromide. For the confirmation of the primary result, a method of reversed probe hybridization, using a nylon membrane immobilized with the oligonucleotide probe specific to the target sequence, was established. The oligonucleotide probe was given a homopolymer tail with terminal deoxyribonucleotidyl-transferase; the tail was spotted onto a nylon membrane and bound covalently by UV irradiation. Owing to its length, the tail bound to the nylon, leaving the oligonucleotide probe free to hybridize. Hybridization of the amplified target sequence to the immobilized probe was accomplished by a simple colorimetric reaction involving the enzymatic oxidation of a colorless chromogen that yielded a purple color wherever hybridization occurred.

Base Sequence↗

PubMed1998

[Comparative analysis of the VP2 variable region of the gene from infectious bursal disease virus isolates].

Variable cDNA regions in the VP2 gene of 24 isolates of infectious bursal disease virus (IBDV) isolated in Russia in 1993-1996 were amplified by the "nested" PCR and sequenced. The primary structure analysis of the VP2 gene variable region revealed 2 major groups of IBDV isolates. The first group consisted of the isolates with the structure identical or closely related to the highly virulent European strains CS89, 74/89A, 661, JY86, and DV86, the second group included the isolates with a high level of homology to the vaccine strains PBG98 and Cu-1. In addition, two isolates with original structure were identified, which differed from previously studied strains.

Amino Acid Sequence↗

PubMed1997

Molecular characterisation of the pifC gene encoding translation initiation factor 3, which is required for normal photosynthetic complex formation in Rhodobacter sphaeroides NCIB 8253.

In order to determine whether translation initiation events play a selective role in regulating the expression of photosynthetic complexes in the photosynthetic bacterium Rhodobacter sphaeroides, we have undertaken an initial study to investigate the potential role of translation initiation factor IF3, which also behaves as a pleiotropic regulatory factor in some bacteria. Following the isolation and purification of a 24-kDa IF3-like protein (PifC) from R. sphaeroides, we used nested PCR to clone and characterise the encoding gene, pifC (photosynthesis-affecting initiation factor). The 545-bp pifC encodes a protein exhibiting 60% identity (78.6% similarity) with the Escherichia coli IF3 (InfC) protein and, in common with all other IF3 genes identified to date, pifC possesses a rare initiation codon (AUA). Furthermore, in common with IF3, PifC was shown here to perform a discriminatory function towards CUG start codons, confirming its role and function as an IF3 in R. sphaeroides. Insertion of a kanamycin resistance cassette into the 5' end of pifC resulted in a viable phenotype which exhibits growth rates similar to wild type but which possesses reduced bacteriochlorophyll and photosynthetic complexes in semi-aerobic cultures. It is shown here that the mutant is still able to produce a PifC protein but that it possesses reduced IF3 activity. This may account for the viable nature of the mutant strain, and may indicate that the effect of the mutation on photosynthesis can be more severe than shown in the present study. The mechanisms by which PifC may exert its selective regulatory effect on photosynthesis expression are discussed.

Amino Acid Sequence↗

PubMed2003

Phylogeny of cooperatively breeding cuckoos (Cuculidae, Crotophaginae) based on mitochondrial gene sequences.

The Crotophaginae is a subfamily of New World cuckoos comprising the monotypic genus Guira and three ani species ( Crotophaga). All exhibit a rare form of cooperative breeding known as plural female joint-nesting, whereby two or more females lay eggs in a single nest. I reconstructed the phylogeny of Crotophaginae using the mitochondrial genes cytochrome oxidase I, II, and III, ATPase 6 and 8, and cytochrome b. The subfamily was monophyletic, implying a single origin of cooperative breeding in New World cuckoos. Crotophaga was also monophyletic with Guira as its sister taxon. Within Crotophaga, the smooth-billed ( C. ani) and groove-billed ( C. sulcirostris) anis formed the internal clade with the greater ani ( C. major) basal to this pair. This phylogeny is consistent with differences in reproductive patterns and social organization exhibited by crotophagine cuckoos, and will serve as a framework for future study of the evolution of cooperative breeding in this subfamily.

Adenosine Triphosphatases↗