Topic
Multiplex polymerase chain reaction
About: Multiplex polymerase chain reaction is a research topic. Over the lifetime, 6409 publications have been published within this topic receiving 221244 citations.
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TL;DR: Having been validated using a panel of well-defined samples from carnivores with known infection status, this approach proved to be useful for the identification of taeniid eggs from both individual animals and for epidemiological studies.
Abstract: A multiplex polymerase chain reaction (PCR) was evaluated for the identification of morphologically indistinguishable eggs of the taeniid tapeworms from carnivores using primers targeting mitochondrial genes. The primers for Echinococcus multilocularis (amplicon size 395 bp) were species-specific as assessed by in silica analysis and in the PCR using well-defined control samples. The design of primers that specifically amplify DNA from E. granulosus or Taenia spp. was not possible. The primers designed for E. granulosus also amplified DNA (117 bp) from E. vogeli, and those designed for Taenia spp. amplified products (267 bp) from species of Mesocestoides, Dipylidium and Diphyllobothrium. Nevertheless, as our diagnostic approach includes the concentration of taeniid eggs by sequential sieving and flotation, followed by their morphological detection, this non-specificity has limited practical importance. Sequence analysis of the corresponding amplicon can identify most of the described E. granulosus genotypes. Taenia spp. can be identified by direct sequencing of the 267 bp amplicon, or, for most species, by restriction fragment length polymorphism (RFLP) analysis. The multiplex PCR was readily able to detect 1 egg (estimated to contain 7000 targets, as determined by quantitative PCR). Having been validated using a panel of well-defined samples from carnivores with known infection status, this approach proved to be useful for the identification of taeniid eggs from both individual animals and for epidemiological studies.
288 citations
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TL;DR: Both multiplex RT‐PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population.
Abstract: There is a need for rapid, sensitive, and accurate diagnosis of lower respiratory tract infections in children, elderly, and immunocompromised patients, who are susceptible to serious complications. The multiplex RT-nested PCR assay has been used widely for simultaneous detection of non-related viruses involved in infectious diseases because of its high specificity and sensitivity. A new multiplex RT-PCR assay is described in this report. This approach includes nested primer sets targeted to conserve regions of human parainfluenza virus haemagglutinin, human coronavirus spike protein, and human enterovirus and rhinovirus polyprotein genes. It permits rapid, sensitive, and simultaneous detection and typing of the four types of parainfluenza viruses (1, 2, 3, 4AB), human coronavirus 229E and OC43, and the generic detection of enteroviruses and rhinoviruses. The testing of 201 clinical specimens with this multiplex assay along with other one formerly described by our group to simultaneously detect and type the influenza viruses, respiratory syncytial viruses, and a generic detection of all serotypes of adenovirus, covers the detection of most viruses causing respiratory infectious disease in humans. The results obtained were compared with conventional viral culture, immunofluorescence assay, and a third multiplex RT-PCR assay for all human parainfluenza viruses types described previously. In conclusion, both multiplex RT-PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population.
288 citations
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TL;DR: PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.
Abstract: The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.
288 citations
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TL;DR: Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.
Abstract: The adoption of molecular genetic techniques has greatly improved our ability to characterize microbial populations in situ, leading to more accurate assessments of pollutant biodegradation and the impact of remediation technologies. Initially, biases associated with culturing were avoided by using DNA hybridization techniques with probes containing the catabolic gene of interest. Cross-hybridization at low stringency (46) and exclusion of related but not identical genes at high stringency (15, 30, 56) may produce false-positive and false-negative results with environmental samples. PCR amplification with primers based on conserved regions within the gene of interest, however, will permit amplification despite variability in the overall sequence and detect genotypes not detected by hybridization alone. Molecular methods, particularly in quantitative PCR (Q-PCR), continue to advance rapidly. Our laboratory described a competitive Q-PCR technique to enumerate catechol 2,3-dioxygenase genes as a means to evaluate bioremediation of aromatic hydrocarbons at petroleum-contaminated sites (35). Here we present primer sets targeting additional aromatic catabolic genes and the use of real-time PCR for quantification to extend our ability to enumerate genotypes involved in biodegradation of aromatic pollutants.
The two main approaches to Q-PCR are competitive and real-time PCR. In competitive PCR, a comparison of the intensities of the standard and target amplicons allows quantification (42). One limitation of this method is that amplification efficiency of the target and standard are not always equivalent. Furthermore, post-PCR analysis of the products is labor-intensive and not amenable to the large-scale screening of environmental samples. Real-time PCR offers an accurate, sensitive method of quantification without the labor-intensive postamplification analysis and assumptions required by competitive PCR. Detection of product during amplification can be accomplished by several means, including molecular beacons (55), hybridization probes (60), and BODIPY FL-labeled probes and primers (27), but hydrolysis probes and DNA-binding dyes are the most commonly reported methods. The hydrolysis probe (TaqMan) assay takes advantage of the 5′-nuclease ability of DNA polymerase to hydrolyze a labeled probe bound to its target amplicon to produce a signal. The SYBR green assay relies on fluorescence signal produced as the dye binds to double-stranded DNA during the extension step. While the hydrolysis probe provides an additional degree of specificity, identifying an additional highly conserved region for the probe may not always be possible. Furthermore, both the probe and the primers must be from a highly conserved region of the target to avoid biasing quantitation (2). Simpler methods using DNA-staining dyes, such as SYBR green I, have been successful (36, 62), do not require the design of an internal hybridization probe, and thus can be used with any established PCR primers with only minor modifications of the described protocols.
This study was conducted to develop multiplex and real-time PCR procedures to quantify aromatic catabolic genes in environmental samples and eventually improve our understanding of biodegradation in the field. The large subunit of aromatic oxygenase genes was chosen as the indicator gene, because it has been implicated in substrate specificity and is one of the rate-limiting steps in aromatic hydrocarbon biodegradation (14) and its DNA sequence is conserved for oxygenases targeting the same substrate. Alignments were constructed from groups of related oxygenase genes, and each primer set was chosen from a conserved region unique to that group of oxygenases. Thus, a single primer set will detect an entire subfamily of related oxygenase genes rather than a species-specific catabolic gene. In all, PCR primer sets which targeted biphenyl dioxygenase, naphthalene dioxygenase, toluene dioxygenase, toluene/xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. Testing and optimization with genetically well-characterized bacterial strains demonstrated the specificity of each primer set. Multiplex PCR protocols were developed to permit simultaneous detection of aromatic oxygenase genes and facilitate rapid screening of environmental samples. Real-time PCR with SYBR green I was used to determine the gene copy number with a quantification limit of 2 × 102 copies of target per reaction mixture. The primer sets and real-time PCR methods presented will be used to assess natural attenuation, investigate the ecology of contaminated sites, and aid in optimization of bioremediation in the field.
287 citations
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TL;DR: The real-time PCR assay was able to detect all MβL-harboring clinical isolates, and the Tm-assigned genotypes were 100% coincident with previous sequencing results, meaning this assay could be suitable for identification of M βL-producing gram-negative bacteria by molecular diagnostic laboratories.
Abstract: Metallo-β-lactamase enzymes (MβL) are encoded by transferable genes, which appear to spread rapidly among gram-negative bacteria The objective of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding MβL-type enzymes based on the amplicon melting peak The reference sequences of all genes encoding IMP and VIM types, SPM-1, GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing different sizes and melting peak temperatures (Tm) The real-time PCR assay was able to detect all MβL-harboring clinical isolates, and the Tm-assigned genotypes were 100% coincident with previous sequencing results This assay could be suitable for identification of MβL-producing gram-negative bacteria by molecular diagnostic laboratories
285 citations