Abstract:
:Development of rapid amplification assays for the detection and identification of biological threat agents has become a focus of diagnostic efforts in recent years. The use of real-time PCR assays as diagnostic tools depends upon two critical processes. First, nucleic acid purification must provide template that is both amplifiable and free of PCR inhibitors. Second, the assays themselves must be sensitive and specific for their nucleic acid targets. A differentiation must be made between results achieved due to the lack of target nucleic acid (true negatives) and those produced due to the inability to amplify target DNA (false negatives) so confidence in negative reactions is possible. False negatives can occur when inhibitors are present in the sample being tested, especially if clinical samples such as blood are analyzed. To address the problem of detecting inhibition in purified nucleic acids, an exogenous internal positive control (IPC) based on Taqman chemistry was developed. A previously optimized assay was cloned and the primer and probe sites were mutated to produce novel sequences with no known homology to published sequence data. The IPC was sensitive to a variety of inhibitors, including hemoglobin, heparin, EDTA, humic acids, and fulvic acid. It was also equally sensitive to inhibition when labeled with either 6FAM or ROX dyes. In addition, the IPC was successfully multiplexed with agent specific assays without any loss in their sensitivity. The designed IPC assay has proven to be an effective tool for monitoring inhibitors of PCR and builds confidence in negative results obtained with agent specific assays.
journal_name
Mol Cell Probesjournal_title
Molecular and cellular probesauthors
Hartman LJ,Coyne SR,Norwood DAdoi
10.1016/j.mcp.2004.07.006subject
Has Abstractpub_date
2005-02-01 00:00:00pages
51-9issue
1eissn
0890-8508issn
1096-1194pii
S0890-8508(04)00073-8journal_volume
19pub_type
杂志文章abstract::The phenotypic detection of Campylobacter concisus, a species of considerable genomic and phenotypic heterogeneity, has proven to be rather tedious in the past. Although alternative methods like DNA:DNA hybridization, immunotyping or whole-cell protein electrophoresis are valuable for the specific detection of C. conc...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/s0890-8508(95)90114-0
更新日期:1995-08-01 00:00:00
abstract::Human cytomegalovirus (HCMV) is responsible for severe infections in immunocompromised patients. Viral load has recently been identified as one of the major risk factors for subsequent development of HCMV disease. In this context, we developed a protocol allowing rapid, sensitive and precise quantification of HCMV DNA...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1006/mcpr.1996.0071
更新日期:1997-02-01 00:00:00
abstract::Many highly homologous genes are present in the murine major histocompatibility complex (MHC) class I gene family. Consequently, it is difficult to distinguish between RNA transcripts of individual class I genes solely on the basis of nucleic acid hybridization analysis using DNA probes over 50 base pairs long. To avo...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/0890-8508(87)90034-x
更新日期:1987-09-01 00:00:00
abstract::We have applied the temperature-dependent single-stranded conformational polymorphism (SSCP) analysis for characterization of influenza A virus cDNA fragments. A series of primers were synthesized on the basis of the comparison of hemagglutinin and PB2 gene sequences of different origin. RT-PCR reactions were run usin...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2008.09.003
更新日期:2008-10-01 00:00:00
abstract::Both Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) can cause high mortality and morbidity in Muscovy ducklings. MDPVs and GPVs share high nucleotide identity, which can cause errors during differential diagnosis. In this study, the NS genes of both MDPVs and GPVs were chosen for the design of specific prim...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2019.101439
更新日期:2019-10-01 00:00:00
abstract::We report the occurrence of a common five-nucleotide deletion at codon 1309 of the adenomatous polyposis coli (APC) gene in four different South African population groups. The mutation causes familial adenomatous polyposis (FAP) in 18% (4/22 unrelated patients screened) of affected South Africans, which is similar to ...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/s0890-8508(95)91007-7
更新日期:1995-02-01 00:00:00
abstract::A multiplex PCR was developed that is capable of detecting four of the most important bacterial agents of atypical pneumonia, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in uncultured patient specimens. These organisms cause similar symptomologies and are often not...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2005.05.002
更新日期:2005-10-01 00:00:00
abstract::The ligase detection reaction (LDR) is a highly specific genotyping method for single nucleotide variations. Although LDR typically discriminates single nucleotide polymorphism (SNP) alleles at the 3' end of so-called LDR discriminating probes, we designed probes in which the position of nucleotide differences for dis...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2010.08.005
更新日期:2010-12-01 00:00:00
abstract:BACKGROUND:Osteoarthritis (OA) is a frequent and incurable joint disease, inducing significant pain and seriously threatening to human health. It has been reported that microRNAs (miRNAs) play crucial roles on cancers and inflammatory diseases via cooperating with genes. However, the effect of miR-374a-3p/Wingless-type...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2020.101541
更新日期:2020-06-01 00:00:00
abstract::The hydroxymethylbilane synthase (HMBS) mRNAs from 44 control individuals and 30 patients suffering from acute intermittent porphyria (AIP), were screened for length differences by reverse transcriptase polymerase chain reaction (RT-PCR) and any abnormalities were characterized by direct sequencing. Examination of the...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1006/mcpr.1997.0153
更新日期:1998-04-01 00:00:00
abstract::Variation amongst strains of Helicobacter pylori and Helicobacter mustelae was examined by DNA restriction endonuclease digestion and rRNA gene patterns generated using a non-radioactive probe. Chromosomal DNA was extracted from 30 cultures of H. pylori from human, Rhesus monkey and pig gastric mucosa, and from three ...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/0890-8508(90)90023-s
更新日期:1990-08-01 00:00:00
abstract::The molecular tag vaccine against Brucella abortus and serological testing are the main methods of prevention of brucellosis used currently. They can discriminate vaccinated animals and humans from those naturally infected. In this study, we constructed a gene deletion mutant strain, B. abortus S19 virB5 with a molecu...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2012.02.003
更新日期:2012-06-01 00:00:00
abstract::DNA samples of 2303 individuals from nine different population groups were screened for variant -175g-->t in the promoter region of the low-density lipoprotein receptor (LDLR) gene. The -175g-->t variant detected at carrier frequencies of 3-10% in different African population groups was absent in the Caucasian and Asi...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/s0890-8508(03)00050-1
更新日期:2003-08-01 00:00:00
abstract::The unique ability of homopyrimidine peptide nucleic acid (PNA) to strand invade homopurine sites of duplex DNA offers a potential alternative to existing techniques for rapid detection of PCR products. From gel shift studies, PNA was found to specifically strand invade homopurine sites that had been incorporated into...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1006/mcpr.2000.0311
更新日期:2000-10-01 00:00:00
abstract::Several dyes are now known which intercalate into double-stranded DNA with fluorescence enhancement. One such is TOTO (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-meth yl-2,3- dihydro-(benzo-1,3-thiazole)-2-methlyidene]-quinolinium tetraiodide), a homodimer of thiazole orange, whose complexes with DNA...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1006/mcpr.1994.1035
更新日期:1994-06-01 00:00:00
abstract::PCR detection and quantification of vaginal lactobacilli remains problematic because of the high level of genetic heterogeneity and taxonomic complexity within the genus Lactobacillus. The aim of the present study was to identify conserved sequences among the genomes of major species of vaginal lactobacilli that could...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2016.11.006
更新日期:2017-04-01 00:00:00
abstract::Multiplex ligation dependent probe amplification (MLPA) assays were designed for the genes HEXB (OMIM: 606873), GM2A (OMIM: 613109) and SMARCAL1 (OMIM: 606622) of humans. Two sets of synthetic MLPA probes for these coding exons were tested. Changes in copy numbers were detected as well as single nucleotide polymorphis...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2012.08.007
更新日期:2013-02-01 00:00:00
abstract::A nucleic acid hybridization assay for the detection of the pilin gene of Neisseria gonorrhoeae has been devised. The method involves solution hybridization of pilin specific synthetic oligonucleotide probes to genomic DNA in crude cell lysates. This is followed by capture of the probe-target complex onto a microtitre...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/0890-8508(89)90037-6
更新日期:1989-03-01 00:00:00
abstract::Human metapneumovirus (hMPV) is a prevalent pathogen worldwide and causes various respiratory infections. Although it is a critical pathogen in pediatric patients, it is unclear how it enters host cells. In this study, we focused on hMPV cell entry using two kinds of cell lines (Vero E6 and LLC-MK2), which are most co...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2016.06.003
更新日期:2016-08-01 00:00:00
abstract::Characterization of antimicrobial resistance and virulence gene profiles provides important information on the potential pathogenicity of bacteria. This information can be used to facilitate prompt and effective treatment of bacterial infections. We developed and tested a PCR-based microarray platform for detecting vi...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2004.11.008
更新日期:2005-06-01 00:00:00
abstract::In a previous study, we reported the performance of a PCR assay amplifying 287-bp of the 16S rRNA gene of thermo-tolerant Campylobacter (C. jejuni, C. lari, C. coli) through an international ring-trial involving 12 participating laboratories. Based on the validated set of primers, a LightCycler real-time PCR assay (LC...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2004.04.005
更新日期:2004-10-01 00:00:00
abstract::A DNA amplification procedure using heat stable Taq polymerase and the polymerase chain reaction is described for the detection of Pseudomonas aeruginosa in specimens from cystic fibrosis patients. A set of primers was selected on the basis of the nucleotide sequence of the algD gene encoding GDP mannose dehydrogenase...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/0890-8508(92)90005-i
更新日期:1992-08-01 00:00:00
abstract::This study reports a significant up-regulation of bta-miR-146a and bta-miR-146b expression levels in bovine mammary tissues infected with subclinical, clinical and experimental mastitis. Potential target genes are involved in multiple immunological pathways. These results suggest a regulatory function of both miRNAs f...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2016.08.004
更新日期:2016-10-01 00:00:00
abstract::The major outer-membrane protein (MOMP) from Campylobacter jejuni NCTC 11168 was purified by solubilization in Triton X-100. Whole-cell proteins of Campylobacter species and the MOMP were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, immunoblotting on nitrocellulose paper and enzyme-linke...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/0890-8508(87)90038-7
更新日期:1987-09-01 00:00:00
abstract::An example of the application and contruction of a polymerase chain reaction (PCR) internal control is presented. The internal control is synthesized in one PCR reaction. The primers used in this reaction possess 5' over-hanging ends which are identical to the primers used in the diagnostic reaction, whereas their 3' ...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1006/mcpr.1998.0170
更新日期:1998-10-01 00:00:00
abstract::Porcine circovirus 3 (PCV3), as a newly emerged circovirus, is widely distributed in pig populations worldwide. Co-infection of PCV2 and PCV3 has been reported frequently in clinical samples. In the present study, a TB Green II-based duplex real-time polymerase chain reaction (qPCR) was developed to rapidly and differ...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2019.04.001
更新日期:2019-06-01 00:00:00
abstract::Human infection with the parasitic nematode Enterobius vermicularis occurs worldwide, particularly in children. Although its prevalence may exceed 35% in some parts of the world, molecular studies of E. vermicularis in humans are limited. The aim of the present study was to investigate the genetic variation within E. ...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2011.03.005
更新日期:2011-04-01 00:00:00
abstract::A polymerase chain reaction (PCR) protocol was developed that could specifically amplify a 520-bp region of the erythromycin resistant methylase (ermC) gene sequence. The identity of the PCR-amplified 520-bp DNA was confirmed by HinCII endonuclease restriction digestion, which produced the predicted 440-bp and 80-bp D...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1006/mcpr.1997.0121
更新日期:1997-10-01 00:00:00
abstract::We developed a completely homogeneous duplex loop-mediated isothermal amplification (LAMP) method. The present LAMP method employed a combination of a 6-carboxyfluorescein (FAM)-labeled primer (donor) for one target gene, a non-labeled primer for the other, and an intercalator ethidium bromide (EtBr) dye (acceptor) on...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1016/j.mcp.2010.03.001
更新日期:2010-08-01 00:00:00
abstract::The aim of this study was to determine the presence of haematogenous neoplastic cells in patients with prostate cancer. Circulating prostate cells can be detected in cancer patients by using a nested-reverse transcriptase-polymerase chain reaction assay (RT-PCR), for prostate-specific membrane (PSM) antigen mRNA. This...
journal_title:Molecular and cellular probes
pub_type: 杂志文章
doi:10.1006/mcpr.1998.0201
更新日期:1998-12-01 00:00:00