Multiple Polymerase Chain Reaction Products Research Articles

Detection of DNA virus in respiratory samples of children by multiplex PCR combined with RDB

Objective To develop multiplex polymerase chain reaction (PCR) combined with reversedotblothy bridization (RDB) method for detection of DNA virus in respiratory samples, and provide a surveillance and rapid diagnosis tool of acute viral respiratory infection. Methods We designed multiple PCR primers and the probes referenced to virus nucleic acid sequences in the National Center for Biotechnology Information (NCBI) database, and fixed specific oligonucleotide probes on the nylon membrane. After multiple PCR amplification of virus DNA of human bocavirus (hBOV), karolinska Institutet (KI), adenovirus (AdV), Washington University polyomaviros (WUPyV), and human parvovirus B19 (HPVB19), the denaturalized amplification products were hybridized with various specific probes, followed by visualization and analysis of the results. The sensitivity and specificity were tested. At the same time, 108 cases of clinical specimens of multiple PCR products were analyzed by reverse spot hybridization detection, and compared to the results of culture method. Results The specific probes of multiple PCR-RDB only hybridized with corresponding amplification products without cross-hybridization reaction with other pathogen. The sensitivity of RDB hybridization was 1 colony-forming units (CFU). The positive rate of 34.26% (37 cases out of 108 cases) with PCR-RDB method was significantly higher than that 27.78% (30 cases out of 108 cases) with common test method. Conclusions The multiplex PCR combined with RDB might become a rapid and simple method to detect the DNA virus in respiratory samples, which might be a promising tool for clinical application. Key words: DNA viruses/isolation & purification; DNA viruses/genetics; DNA viruses/pathogenicity; Respiratory tract infections/virology; Respiratory tract infections/genetics; Polymerase chain reaction; Gene amplification; Nucleic acid probes; Child

The accuracy of Influenza A (H1N1) “swine flu” laboratory testing: A systematic review of diagnostic test accuracy.

The accuracy of Influenza A (H1N1) “swine flu” laboratory testing: A systematic review of diagnostic test accuracy.

Report From the Jerusalem Workshop on Lynch Syndrome-Hereditary Nonpolyposis Colorectal Cancer

A Workshop was held in Jerusalem, Israel, on October 26 and 27, 2009 to discuss the management of Lynch syndrome-hereditary nonpolyposis colorectal cancer (CRC), with the primary goal to develop consensus for the optimal management of this disease. A second goal was to identify areas of research with the potential to advance the clinical management of Lynch syndrome. The perspectives and recommendations from the workshop are meant to be a platform for discussion and deliberation. The Workshop was organized by Moshe Shike (Memorial Sloan Kettering Cancer Center, New York) and sponsored by The Colon Cancer Foundation. More details of each presentation are available in an on-line supplement.

The accuracy of Influenza A (H1N1) “swine flu” laboratory testing: A systematic review of diagnostic test accuracy.

The accuracy of Influenza A (H1N1) “swine flu” laboratory testing: A systematic review of diagnostic test accuracy.

Identification of novel human leukocyte antigen G alleles in an East African population by high-resolution sequence-based typing

We report two novel human leukocyte antigen G (HLA-G) alleles identified in an East African population during sequence-based typing of HLA-G. The novel alleles were confirmed by sequencing multiple polymerase chain reaction products and molecular cloning and subsequent sequencing of multiple clones. The sequence of HLA-G*0110 (EU290672) is identical to G*01010101/01010102/01010103/01010104/01010105 at exons 2, 3, and 4 except for a single nucleotide difference at codon 31 (ACG --> ATG), resulting in a coding change from threonine to methionine. The sequence of HLA-G*0111 (EU290673) is identical to G*010404 at exons 2, 3, and 4 except for a single nucleotide difference at codon 31 (ACG --> ATG), resulting in a coding change from threonine to methionine. These new alleles are detected in several other individuals in our study population and the functional relevance of these new alleles must be studied.

Determination of Genetically Modified Soybean by Multiplex PCR and CGE with LIF Detection

A method for rapid screening, identification and detection of genetically modified soybean by multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) was developed and applied to actual food samples. A triplex PCR procedure was used to amplify the parts of nopaline synthase (NOS) terminator, and the junction between cauliflower mosaic virus 35S promoter and chloroplast transit peptide CTP4 trait gene, as well as the lectin gene to allow the screening and identification of specific transgenic soybean line (glyphosate-tolerant soybean). The multiplex PCR parameters and conditions of capillary gel electrophoresis were optimized. The amplified DNA fragments were analyzed by CGE–LIF. The amplified PCR products were analyzed by CGE–LIF within about 20 min. The method developed is highly sensitive and allows the detection of a percentage of genetically modified soybean as low as 0.025%. The percentage is low enough to fulfill the requirement of the EU Regulation for transgenic food labeling of 1.0%. The sequences of the multiple PCR products were identical with those published in Genbank. The proposed method has been used in identification and detection of genetically modified soybean in various food samples. Compared with agarose gel electrophoresis (AGE), the proposed method is more rapid, accurate and requires a smaller amount of samples. Thus an efficient alternative method is provided for monitoring genetically modified soybean in order to meet the increasing demand of implementation of the genetically modified food labeling policy.

DNA probe chip system for multiple detection of food poisoning microorganisms

A DNA probe chip system that can simultaneously analyze multiple genes at the place where a specimen is offered has been developed. Genes to be detected were extracted from food-contaminating microorganisms ( Escherchia coli O157:H7 and Salmonella typhimurium in this study) and then amplified by multiplex polymerase chain reaction (PCR) (utilizing the reverse primers with fluorescein at each 5′-end). These DNA products were measured in a chip system containing capture probes specific to the respective product. The probes were synthesized to contain biotin at each 5′-end and bound onto spatially separated glass surfaces via biotin–streptavidin linkage. Upon addition of a PCR mixture after denaturation, hybridization between the capture probe and the DNA product occurred, which was sequentially monitored utilizing an immune reaction network. Provision of such simplified analytical protocol for multiple PCR products was achieved without a significant sacrifice in the detection sensitivities.

Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions

DNA sequencing is considered the gold standard for nucleic acid identification and mutation detection. However, sequencing is labor intensive because it requires previous amplification and only a single sequence is analyzed at a time. We developed two novel strategies that substantially improve DNA sequencing. The first allows multiple polymerase chain reaction (PCR) products to be sequenced in a single sequencing reaction and analyzed simultaneously in a single lane or capillary. Simultaneous sequencing by this method, designated "SimulSeq," can provide either simultaneous single-direction sequencing of multiple genes or simultaneous forward and reverse sequencing from a single gene. In the second approach, designated "AmpliSeq," we demonstrate a technique combining PCR amplification and sequencing in a single reaction that is analyzed in a single lane or capillary. We demonstrate combined PCR with short bidirectional sequencing, and combined PCR with unidirectional sequencing. We anticipate that these methods will have utility in research and clinical settings where panels of mutations or large numbers of samples are be analyzed and/or when turnaround time is critical.

In-frame cloning of synthetic genes using PCR inserts.

Because many genes of biological interest are larger than the maximum size that current synthetic oligonucleotide synthesizers can produce (approx 110 bases), there is a need for methods that allow rapid production and expression of genes constructed from multiple synthetic DNA fragments. A number of synthetic genes have been generated using the recursive polymerase chain reaction (PCR) method, but problems concerning primer design and incorrect final gene sequence, especially with large synthetic genes, are a concern (-). The cloning method described here follows a series of steps in which multiple PCR products or synthetic duplex oligonucleotides are positionally cloned into a plasmid vector (). A synthetic gene of practically any sequence or length can be built using the in-frame cloning method. Genes are assembled such that open reading frames are maintained by linking DNA fragments through the use of six basepair blunt-end restriction sites. Each cloning step uses an anchored sticky-end restriction site and a variable blunt-end restriction site that result in specific insert orientation and high cloning efficiencies. The overall strategy of in-frame cloning allows the researcher total control over nucleotide sequence, codon usage, promoter and other regulatory elements, and the placement of unique restriction sites throughout the recombinant construct. One advantage of the in-frame cloning method described here is that it allows for flexible yet precise construction of synthetic genes using standard recombinant techniques. Another advantage is that it employs inexpensive, readily available materials.

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers.

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3'-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.

Variable R1 region in varicella zoster virus in fulminant type of acute retinal necrosis syndrome

BACKGROUND/AIMSVaricella zoster virus (VZV) is a causative agent in acute retinal necrosis (ARN) syndrome. However, in spite of aggressive antiviral therapy, clinical characteristics among patients have varied. Different viral strains...

An actin gene-related polymerase chain reaction (PCR) test for identification of chicken in meat mixtures

Using the polymerase chain reaction (PCR) and DNA extracted from muscle, a single pair of oligonucleotide primers can yield amplification products from several members of the actin multigene family simultaneously. These multiple PCR products form species-specific “fingerprints” on gel electrophoresis which may be useful for meat authentication. However, for analysis of meat mixtures, the presence of a single band unique to a species would have many advantages over a multi-component fingerprint. A procedure is described in which primers amplify at a single actin gene locus, giving a positive band with DNA extracted from chicken and turkey, but no reaction with duck, pheasant, porcine, bovine, ovine or equine DNA. The chicken signal was clearly detectable with DNA from meat admixtures containing 1% chicken/99% lamb and from meat heat-treated at 120°C. For further discrimination, the chicken PCR product could be differentiated from turkey by restriction enzyme digestion.

Efficiency of Randomly Amplified Polymorphic DNA to Sequence Characterized Amplified Region Marker Conversion and their Comparative Polymerase Chain Reaction Sensitivity in Cucumber

The conversion of randomly amplified polymorphic DNA (RAPD) markers to sequence characterized amplified region (SCAR) markers, and the effects of differing polymerase chain reaction (PCR) conditions were studied in cucumber (Cucumis sativus L.). Attempts were made to clone and sequence 75 RAPD PCR products to produce SCAR primers (16 to 22 nucleotides) designed to amplify original RAPD PCR products. The influence of template DNA source, purity, and concentration, MgCl2 concentration, Taq polymerase source, and type of thermocycler upon RAPD and SCAR marker performance was evaluated. Conversion of RAPD to SCAR markers was not universally successful, and SCAR primers reacted differently to varying PCR conditions. Only 48 (64%) of 75 RAPD markers were successfully converted to SCAR markers and 11 (15%) of these reproduced the polymorphism observed with the original RAPD PCR product. Moreover, some SCAR primer pairs produced multiple polymorphic PCR products. The band intensity of SCAR markers were brighter (P = 0.05) than their corresponding RAPD markers with only one exception. The SCAR markers examined were less influenced (P = 0.05) by MgCl2 concentration than their corresponding RAPD markers. However, some SCAR markers were more sensitive to reaction impurities than their RAPD counterparts and SCAR markers tended to be less readily visualized (decrease in frequency of visible PCR product) with low concentrations (1 and 2 mm) of template DNA than their corresponding RAPD markers. Neither the source of Taq nor the type of thermocycler used affected the performance of SCAR and RAPD markers. These data suggest that although SCAR markers may demonstrate enhanced performance over the RAPD markers from which they are derived, careful consideration must be given to both the costs and potential benefits of SCAR marker development in cucumber.

Genetic Characterization and Phylogeny of Sabiá Virus, an Emergent Pathogen in Brazil

Sabiá virus, one of five arenaviruses from South America known to cause hemorrhagic fever in humans, emerged in 1990 when it was isolated from a fatal case in Sao Paulo, Brazil. Subsequently, it has caused two laboratory-acquired infections. Its natural distribution and host are still unknown. Using viral RNA and multiple polymerase chain reaction products as templates, the nucleotide sequence of the small (S) RNA segment of Sabiá virus, which codes for the nucleocapsid (N) and glycoprotein precursor, was determined. This virus shares an ambisense genome in common with other arenaviruses, although it has a unique predicted three stem–loop structure in the S RNA intergenic region. Phylogenetic analysis of a portion of the N gene sequence confirmed that Sabiá virus is distinct from all other members of the Arenaviridae and shares a progenitor with Junin, Machupo, Tacaribe, and Guanarito viruses.

Low hprt mRNA levels and multiple hprt mRNA species in 6-thioguanine-resistant Chinese hamster cell mutants possessing nonsense mutations

Previous studies suggested that many Chinese hamster ovary (CHO) cell hprt mutants having point mutations in the protein coding region also have low steady-state hprt mRNA concentrations. In addition, polymerase-chain reaction (PCR) amplification of hprt cDNA synthesized from some of these mutants results in multiple products containing deleted exons indicating that these mutants posses multiple species of hprt mRNA. In this study, we have used northern blot analysis to quantify the concentrations of hprt mRNA in 86 mutants known to possess point mutations leading to either missense or nonsense mutations. 28 of 35 nonsense mutants (80%), but only 7 of 51 missense mutants (14%), had < 50% of the hprt mRNA concentration found in parental CHO cells. Furthermore, all the nonsense mutants with premature termination codons in the internal exons of the gene ( i.e., exons 3, 4, 5, 6 and 7) showed a significant reduction (averages < 16% of parental) in the steady-state levels of hprt mRNA, while nonsense mutants with termination codons situated in the extreme 5′ and 3′ regions of the gene had near parental hprt mRNA levels. In the same collection of mutants, the proportion of mutants producing multiple cDNA PCR products was much greater ( 18 35 ) for mutants having nonsense mutations than for mutants with missense mutations ( 2 51 ). All nonsense mutants with mutations in exons 3, 4 and 5 produced multiple species, while all those with mutations in exons 7, 8 and 9 produced a single PCR product. These results suggest that sequence changes in mammalian genes that affect protein chain length can also affect mRNA concentration and the splicing of pre-mRNA molecules.

DNA sequence analysis of 1-nitrosopyrene-induced mutations in the hprt gene of Chinese hamster ovary cells.

DNA sequence was determined in 21 mutants induced at the hprt locus of Chinese hamster ovary (CHO) cells by 1-nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene. Following cDNA synthesis using RNA from each of the mutants, the hprt protein-coding region was amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequence analysis. Sixteen primary mutations were found: seven were G:C----T:A transversions, five were G:C----A:T transitions, two were single basepair insertions, one was a single basepair deletion, and one was a complex mutation involving substitutions at two A:T basepairs. The simple basepair substitution mutations preferentially occurred with one or two purines 3' to the mutated dG, and mutations in exons 1-4 disproportionately occurred with the mutated dG on the nontranscribed DNA strand. In addition, 12 of the mutants produced one or more cDNA PCR products with partial or complete exon deletions. Seven mutants with multiple PCR products had point mutations in one of the products; exon deletions in the other product(s) removed these point mutations. A group of solvent control mutants had a different distribution of basepair substitution mutations and a lower proportion of cDNAs with exon deletions than that found for the 1-nitrosopyrene-induced mutants. The results indicate a specificity for the induction of mutations in the hprt gene of CHO cells by 1-nitrosopyrene with respect to both the types of mutations produced and their location in the hprt gene. Also, the elimination of point mutations in many of the cDNA PCR products with exon deletions suggests that mutations in the protein-coding sequence affect hprt mRNA processing.