Simple, sensitive, and specific detection of human immunodeficiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers.

Abstract:

:A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for the detection of human immunodeficiency virus type 1 (HIV-1) is described. We have improved all three PCR steps: sample preparation, DNA amplification, and detection of the amplified product. Some of the improvements have been described previously, but they have never been combined into a complete PCR protocol. Peripheral blood mononuclear cells were lysed directly in a buffer containing sodium dodecyl sulfate, Triton X-100, and proteinase K. This crude cell lysate was amplified in a two-step PCR, first with outer primers and then with inner primers nested within the first primers. The PCR product was visualized by agarose gel electrophoresis and ethidium bromide staining. Thus, we avoided conventional DNA extraction as well as hybridization for the detection of the PCR product. The samples were analyzed with four sets of nested primers (JA4 through JA7, JA9 through JA12, JA13 through JA16, and JA17 through JA20) designed to amplify HIV-1 gag, env gp120, env gp41, and pol sequences, respectively. We were able to amplify HIV-1 sequences in all samples from 90 HIV-1-seropositive individuals with mostly mild symptoms. Of these individuals, 24 were negative in HIV-1 isolation and 9 were selected because they were infected by African and Haitian HIV-1 strains. Eighty-five (94%) individuals were positive with at least three of four primer sets. Samples from 26 healthy blood donors, as well as cells infected in vitro with human immunodeficiency virus type 2 and human T-cell leukemia virus type I, were negative in PCR, thus demonstrating the specificity of the amplification.

journal_name

J Clin Microbiol

authors

Albert J,Fenyö EM

doi

10.1128/JCM.28.7.1560-1564.1990

subject

Has Abstract

pub_date

1990-07-01 00:00:00

pages

1560-4

issue

7

eissn

0095-1137

issn

1098-660X

journal_volume

28

pub_type

杂志文章
  • New acquisition of antibiotic-resistant organisms in skilled nursing facilities.

    abstract::The epidemiology of new acquisition of antibiotic-resistant organisms (AROs) in community-based skilled nursing facilities (SNFs) is not well studied. To define the incidence, persistence of, and time to new colonization with methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), a...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.06469-11

    authors: Fisch J,Lansing B,Wang L,Symons K,Cherian K,McNamara S,Mody L

    更新日期:2012-05-01 00:00:00

  • Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries.

    abstract::Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea among children, human immunodeficiency virus-infected patients, and travelers to developing regions of the world. The pathogenesis of EAEC strains involves the production of biofilm. In this study, we deter...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.01128-06

    authors: Mohamed JA,Huang DB,Jiang ZD,DuPont HL,Nataro JP,Belkind-Gerson J,Okhuysen PC

    更新日期:2007-01-01 00:00:00

  • Septic arthritis involving Capnocytophaga ochracea.

    abstract::Septic arthritis of the knee developed in a 21-month-old child. The causative organism, isolated from two separate arthrocenteses, was identified as Capnocytophaga ochracea morphologically and by biochemical reactions. Previous human infections (bacteremias) have occurred in granulocytopenic hosts with concomitant ora...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.19.4.538-540.1984

    authors: Winn RE,Chase WF,Lauderdale PW,McCleskey FK

    更新日期:1984-04-01 00:00:00

  • Evaluation of Staph ID 32 system and Staph-Zym system for identification of coagulase-negative staphylococci.

    abstract::The purpose of the investigation was to evaluate two commercially available identification systems: a new modification of the Staph-Zym system (Rosco, Tåstrup, Denmark) and the Staph ID 32 API system (API System, BioMérieux, Paris, France). A local standard method to be used in routine laboratories was also evaluated....

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.33.5.1150-1153.1995

    authors: Renneberg J,Rieneck K,Gutschik E

    更新日期:1995-05-01 00:00:00

  • Isolation of Bacteroides fragilis from the feces of diarrheic calves and lambs.

    abstract::A selective medium was developed for the isolation of Bacteroides fragilis directly from ovine and bovine fecal samples. The medium (tryptose blood agar plus polymyxin B, triclosan (Irgasan), novobiocin, and nalidixic acid) permitted growth of B. fragilis and several other species of Bacteroides but inhibited growth o...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.21.3.472-473.1985

    authors: Border M,Firehammer BD,Shoop DS,Myers LL

    更新日期:1985-03-01 00:00:00

  • Multilocus sequence typing of Neisseria meningitidis directly from clinical samples and application of the method to the investigation of meningococcal disease case clusters.

    abstract::Infections associated with Neisseria meningitidis are a major public health problem in England, Wales, and Northern Ireland. Currently, over 40% of cases are confirmed directly from clinical specimens using PCR-based methodologies without an organism being isolated. A nested/seminested multilocus sequence typing (MLST...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.43.12.6007-6014.2005

    authors: Birtles A,Hardy K,Gray SJ,Handford S,Kaczmarski EB,Edwards-Jones V,Fox AJ

    更新日期:2005-12-01 00:00:00

  • Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing.

    abstract::We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, ...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/jcm.40.8.2964-2972.2002

    authors: García Del Blanco N,Dobson ME,Vela AI,De La Puente VA,Gutiérrez CB,Hadfield TL,Kuhnert P,Frey J,Domínguez L,Rodríguez Ferri EF

    更新日期:2002-08-01 00:00:00

  • Elimination of Fc receptor binding of human immunoglobulin G in immunofluorescence assays for herpes simplex virus antibodies.

    abstract::The binding of human immunoglobulin G by Fc receptors in herpes simplex virus (HSV)-infected cells can cause false-positive interpretations in the immunofluorescence test for HSV antibody. When the infected cell smears were treated with 10% glacial acetic acid for 5 min and rinsed in phosphate-buffered saline before t...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.24.4.672-674.1986

    authors: Gallo D

    更新日期:1986-10-01 00:00:00

  • Nucleotide sequence-based analysis for determining the molecular epidemiology of Penicillium marneffei.

    abstract::The dimorphic fungus, Penicillium marneffei, is an emerging opportunistic pathogen endemic in Southeast Asia, especially for those with impaired cellular immunity such as human immunodeficiency virus-infected persons. A discriminatory and reproducible method based on the analysis of nucleotide sequences would facilita...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.00567-06

    authors: Lasker BA

    更新日期:2006-09-01 00:00:00

  • Immunodiagnosis of human cysticercosis (Taenia solium) with antigens purified by monoclonal antibodies.

    abstract::Monoclonal antibodies were generated from mice immunized with scolex protein antigen of Cysticercus cellulosae. Three monoclonal antibodies specific for cysticercal antigens, which did not show any cross-reactivity with Taenia solium or Taenia saginata antigens, were selected. Each monoclonal antibody coupled to Sepha...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.25.7.1181-1185.1987

    authors: Nascimento E,Tavares CA,Lopes JD

    更新日期:1987-07-01 00:00:00

  • Detection of Mycobacterium bovis in bovine clinical specimens using real-time fluorescence and fluorescence resonance energy transfer probe rapid-cycle PCR.

    abstract::Nucleic acid sequence capture extraction was coupled with LightCycler PCR amplification and product detection using real-time fluorescence for rapid, definitive detection of Mycobacterium bovis in lymph node specimens from 38 cattle with bovine tuberculosis lesions. PCR amplification of sequence-captured DNA using bot...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.39.4.1272-1278.2001

    authors: Taylor MJ,Hughes MS,Skuce RA,Neill SD

    更新日期:2001-04-01 00:00:00

  • A multidrug-resistant Staphylococcus epidermidis clone (ST2) is an ongoing cause of hospital-acquired infection in a Western Australian hospital.

    abstract::We report the molecular epidemiology of 27 clinical multidrug-resistant Staphylococcus epidermidis (MDRSE) isolates collected between 2003 and 2007 in an Australian teaching hospital. The dominant genotype (sequence type 2 [ST2]) accounted for 85% of the isolates tested and was indistinguishable from an MDRSE genotype...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.06456-11

    authors: Widerström M,McCullough CA,Coombs GW,Monsen T,Christiansen KJ

    更新日期:2012-06-01 00:00:00

  • Comparison of three procedures for biochemical testing of anaerobic bacteria.

    abstract::The Analytab Products, Inc. (API), anaerobic multitest microsystem (MICRO) was compared with the Center for Disease Control conventional (CONV) thioglycolate (supplemented with hemin and vitamin K1) system and with pre-reduced anaerobically sterilized (PRAS) media as recommended by the Virginia Polytechnic Institute. ...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.1.1.15-24.1975

    authors: Moore HB,Sutter VL,Finegold SM

    更新日期:1975-01-01 00:00:00

  • Performance of human immunodeficiency virus type 1 gp41 assays for detecting enfuvirtide (T-20) resistance mutations.

    abstract::The performance of a gp41 assay, created using reagents designed for use with the OpenGene DNA Sequencing System, was evaluated using a panel of plasma samples obtained from enfuvirtide-naive and -experienced human immunodeficiency virus type 1-infected subjects. Resulting sequence data were highly accurate compared t...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.00666-06

    authors: Marcial M,Lu J,Deeks SG,Ziermann R,Kuritzkes DR

    更新日期:2006-09-01 00:00:00

  • Evaluation of two BBL Crystal systems for identification of some clinically important gram-negative bacteria.

    abstract::The BBL Crystal system (Becton Dickinson Microbiology Systems, Cockeysville, Md.) is a miniaturized bacterial identification method employing modified conventional and chromogenic substrates. Two products are currently available, the Rapid Stool/Enteric ID Kit and the Enteric/Nonfermenter ID Kit, each comprising thirt...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.32.9.2221-2224.1994

    authors: Holmes B,Costas M,Thaker T,Stevens M

    更新日期:1994-09-01 00:00:00

  • Evaluation of PCR primers for early diagnosis of cytomegalovirus infection following liver transplantation.

    abstract::The availability of microbiologic methods that detect early replication of cytomegalovirus (CMV) posttransplantation will enhance the process of initiating preemptive antiviral therapy prior to the appearance of CMV disease. Using PCR techniques we sought to determine which region of the CMV genome present in peripher...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.36.2.526-530.1998

    authors: Mendez JC,Espy MJ,Smith TF,Wilson JA,Paya CV

    更新日期:1998-02-01 00:00:00

  • Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum antigens in human fecal specimens using the triage parasite panel enzyme immunoassay.

    abstract::The Triage parasite panel (BIOSITE Diagnostics, San Diego, Calif.) is a new qualitative enzyme immunoassay (EIA) panel for the detection of Giardia lamblia, Entamoeba histolytica/E. dispar, and Cryptosporidium parvum in fresh or fresh, frozen, unfixed human fecal specimens. By using specific antibodies, antigens speci...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.38.9.3337-3340.2000

    authors: Garcia LS,Shimizu RY,Bernard CN

    更新日期:2000-09-01 00:00:00

  • Enzyme immunoassay detecting teichoic and lipoteichoic acids versus cerebrospinal fluid culture and latex agglutination for diagnosis of Streptococcus pneumoniae meningitis.

    abstract::A newly developed enzyme immunoassay (EIA) was used to detect the presence of pneumococcal teichoic and lipoteichoic acids in cerebrospinal fluid (CSF) from patients with Streptococcus pneumoniae meningitis who were being treated with antibiotics. All initial CSF samples, which on culture grew S. pneumoniae, were posi...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.36.8.2346-2348.1998

    authors: Stuertz K,Merx I,Eiffert H,Schmutzhard E,Mäder M,Nau R

    更新日期:1998-08-01 00:00:00

  • A cost-effective approach for detection of toxigenic Clostridium difficile: toxigenic culture using ChromID Clostridium difficile agar.

    abstract::We evaluated the performance and the cost of toxigenic culture using a commercial chromogenic medium (CDIF) for 538 stool specimens. Compared with real-time PCR, this method was found to detect an additional 9% of positive specimens and result in 61% reduction in material costs, with a trade-off increase in turnaround...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.03113-13

    authors: Luk S,To WK,Ng TK,Hui WT,Lee WK,Lau F,Ching AM

    更新日期:2014-02-01 00:00:00

  • Typing and subtyping influenza virus using DNA microarrays and multiplex reverse transcriptase PCR.

    abstract::A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding approximately 500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound m...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.39.2.696-704.2001

    authors: Li J,Chen S,Evans DH

    更新日期:2001-02-01 00:00:00

  • Mechanical homogenization increases bacterial homogeneity in sputum.

    abstract::Sputum obtained from patients with cystic fibrosis (CF) is highly viscous and often heterogeneous in bacterial distribution. Adding dithiothreitol (DTT) is the standard method for liquefaction prior to processing sputum for molecular detection assays. To determine if DTT treatment homogenizes the bacterial distributio...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.00487-14

    authors: Stokell JR,Khan A,Steck TR

    更新日期:2014-07-01 00:00:00

  • Clinical relevance of multiple single-nucleotide polymorphisms in Pneumocystis jirovecii Pneumonia: development of a multiplex PCR-single-base-extension methodology.

    abstract::Pneumocystis jirovecii pneumonia (PcP) is a major cause of respiratory illness in patients with AIDS. The identification of multiple single-nucleotide polymorphisms (SNPs) at three distinct P. jirovecii loci encoding dihydrofolate reductase (DHFR), mitochondrial large-subunit rRNA (mtLSU rRNA), and superoxide dismutas...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.02303-10

    authors: Esteves F,Gaspar J,De Sousa B,Antunes F,Mansinho K,Matos O

    更新日期:2011-05-01 00:00:00

  • Turnover of nontypable Haemophilus influenzae in the nasopharynges of healthy children.

    abstract::The nasopharyngeal Haemophilus influenzae flora of healthy children in a day care center was analyzed by repeated sampling during 4 winter months. The average carrier rate was 39%, but 74% of the children became colonized at some time during the study. The H. influenzae isolates were identified by capsular type, bioty...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.27.10.2175-2179.1989

    authors: Trottier S,Stenberg K,Svanborg-Edén C

    更新日期:1989-10-01 00:00:00

  • Second serogroup of Legionella hackeliae isolated from a patient with pneumonia.

    abstract::A Legionella-like organism (strain 798-PA-H, ATCC 35999) isolated from a lung aspirate was shown by DNA hybridization to belong to the species Legionella hackeliae. Growth, gas-liquid chromatographic, and physiologic characteristics were consistent with those of the type strain of L. hackeliae, Lansing 2 (ATCC 35250)....

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.22.4.488-489.1985

    authors: Wilkinson HW,Thacker WL,Steigerwalt AG,Brenner DJ,Ampel NM,Wing EJ

    更新日期:1985-10-01 00:00:00

  • Comparison of broth microdilution, E Test, and agar dilution methods for antibiotic susceptibility testing of Campylobacter jejuni and Campylobacter coli.

    abstract::A standardized broth microdilution method was compared to the E test and an agar dilution method for the antimicrobial susceptibility testing of Campylobacter jejuni and C. coli isolates. A group of 47 human clinical isolates, 37 isolates from retail poultry, and 29 isolates from living turkeys (total, 113 isolates) w...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/jcm.41.3.1062-1068.2003

    authors: Luber P,Bartelt E,Genschow E,Wagner J,Hahn H

    更新日期:2003-03-01 00:00:00

  • Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene.

    abstract::To differentiate the Borrelia burgdorferi sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial recA gene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplifi...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.38.7.2756-2759.2000

    authors: Pietilä J,He Q,Oksi J,Viljanen MK

    更新日期:2000-07-01 00:00:00

  • Acetylene reduction (dinitrogen fixation) by clinical isolates of Klebsiella pneumoniae.

    abstract::Freshly isolated clinical strains of Klebsiella were tested for the ability to fix dinitrogen by the acetylene reduction assay. Ability to detect this trait was markedly affected by cultural conditions. When the test was run at 37 degrees C in the presence of yeast extract (50 mg/liter), only 1.6% of the organisms wer...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:

    authors: Chambers CA,Silver WS

    更新日期:1977-11-01 00:00:00

  • Antifungal drug susceptibility and phylogenetic diversity among Cryptococcus isolates from dogs and cats in North America.

    abstract::Molecular types of the Cryptococcus neoformans/Cryptococcus gattii species complex that infect dogs and cats differ regionally and with host species. Antifungal drug susceptibility can vary with molecular type, but the susceptibility of Cryptococcus isolates from dogs and cats is largely unknown. Cryptococcus isolates...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.03392-13

    authors: Singer LM,Meyer W,Firacative C,Thompson GR 3rd,Samitz E,Sykes JE

    更新日期:2014-06-01 00:00:00

  • Sonication of explanted prosthetic components in bags for diagnosis of prosthetic joint infection is associated with risk of contamination.

    abstract::Explanted orthopedic implants from 54 patients with aseptic failure and 24 patients with prosthetic knee or hip infection were sonicated in polyethylene bags. The sensitivities of periprosthetic tissue and sonicate fluid cultures for the diagnosis of prosthetic joint infection were 54% and 75%, whereas the specificiti...

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.44.2.628-631.2006

    authors: Trampuz A,Piper KE,Hanssen AD,Osmon DR,Cockerill FR,Steckelberg JM,Patel R

    更新日期:2006-02-01 00:00:00

  • Confirmation of the distinct genotype groups within the form species Candida parapsilosis.

    abstract::The DNA relatedness and restriction fragment length polymorphism patterns of whole-cell DNA and the general phenotypic properties of 14 isolates of the form species Candida parapsilosis representing three diverse genetic groups were compared. The data confirm the unrelatedness of the three groups at the species level....

    journal_title:Journal of clinical microbiology

    pub_type: 杂志文章

    doi:10.1128/JCM.36.1.216-218.1998

    authors: Roy B,Meyer SA

    更新日期:1998-01-01 00:00:00