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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: In this article, the authors demonstrate a fast and easy way of generating standardised DNA templates, which are then used to balance the amplification success for the different targets and to determine the sensitivity of each primer pair in the multiplex PCR.
Abstract: 1. Multiplex PCR is a valuable tool in many biological studies but it is a multifaceted procedure that has to be planned and optimised thoroughly to achieve robust and meaningful results. In particular, primer concentrations have to be adjusted to assure an even amplification of all targeted DNA fragments. Until now, total DNA extracts were used for balancing primer efficiencies; however, the applicability for comparisons between taxa or different multiple-copy genes was limited owing to the unknown number of template molecules present per total DNA. 2. Based on a multiplex system developed to track trophic interactions in high Alpine arthropods, we demonstrate a fast and easy way of generating standardised DNA templates. These were then used to balance the amplification success for the different targets and to subsequently determine the sensitivity of each primer pair in the multiplex PCR. 3. In the current multiplex assay, this approach led to an even amplification success for all seven targeted DNA fragments. Using this balanced multiplex PCR, methodological bias owing to variation in primer efficiency will be avoided when analysing field-derived samples. 4. The approach outlined here allows comparing multiplex PCR sensitivity, independent of the investigated species, genome size or the targeted genes. The application of standardised DNA templates not only makes it possible to optimise primer efficiency within a given multiplex PCR, but it also offers to adjust and/or to compare the sensitivity between different assays. Along with other factors that influence the success of multiplex reactions, and which we discuss here in relation to the presented detection system, the adoption of this approach will allow for direct comparison of multiplex PCR data between systems and studies, enhancing the utility of this assay type.

178 citations

Journal ArticleDOI
TL;DR: The use of tagged primers provides a method for primer design that eliminates the multiple optimization steps involved in developing multiplex PCR.
Abstract: We have developed a simplified method for multiplex PCR based on the use of chimeric primers. Each primer contains a 3' region complementary to sequence-specific recognition sites and a 5' region made up of an unrelated 20-nucleotide sequence. Identical reaction conditions, cycling times, and annealing temperatures have been established for any PCR primer pair comprising the chimeric motif. Under these conditions, efficient multiplex amplification is achieved easily and reproducibly by simple adjustment of the individual primer concentrations. No additional modification of either the reaction components or annealing temperatures is required. The use of tagged primers provides a method for primer design that eliminates the multiple optimization steps involved in developing multiplex PCR.

177 citations

Journal ArticleDOI
TL;DR: The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.
Abstract: Eight pairs of published PCR primers were evaluated for the specific detection of Cryptosporidium parvum and Giardia lamblia in water. Detection sensitivities ranged from 1 to 10 oocysts or cysts for purified preparations and 5 to 50 oocysts or cysts for seeded environmental water samples. Maximum sensitivity was achieved with two successive rounds of amplification and hybridization, with oligonucleotide probes detected by chemiluminescence. Primer annealing temperatures and MgCl2 concentrations were optimized, and the specificities of the primer pairs were determined with closely related species. Some of the primers were species specific, while others were only genus specific. Multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was demonstrated with primers amplifying 256- and 163-bp products from the 18S rRNA gene of Cryptosporidium and the heat shock protein gene of Giardia, respectively. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.

177 citations

Journal ArticleDOI
TL;DR: The multiplex PCR protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.
Abstract: Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were als...

176 citations

Journal ArticleDOI
TL;DR: A modified PCR method which alows walking and sequencing in any direction along genomic DNA starting from a known sequence is developed and successfully walked along the nematode unc 31 gene contained on a YAC within the background of total yeast DNA.
Abstract: Recently, a method named inverse PCR has been described by which genomic DNA sequences adjacent to a known (sequenced) locus can be amplified in vitro and sequenced (1-2). The method comprises the following three steps: (i) restriction of genomic DNA; (ii) recirculanzation of linearised fragments by ligation; and (iii) in vitro amplification with two primers which are reversed in their direction in respect to their normal orientation for PCR. The circularization reaction is unreliable and often concatamers are observed. We have developed a modified PCR method which alows walking and sequencing in any direction along genomic DNA starting from a known sequence. The basic principle of the method is outlined in figure la. There are several advantages of the new method. Ligation of ohgo-cassettes with sticky ends does not cause any problem. Since the cassette contains the universal primer sequence sequencing of PCR products can be done with any of the existing dideoxy termination or chemical degradation techniques using radioor fluorescently-labelled nucleotides or primers. PCR products can easily be sequenced from both ends. The sequence obtained from the specific primer confirms the overlap and the sequence from the universal primer gives the necessary information from the endpoint of the PCR product to design new primers for the next walking/sequencing step. We have successfully walked along the nematode unc 31 gene contained on a YAC within the background of total yeast DNA (figure lb). The technique has also been used to walk along total human DNA from exon 51 of the DMD gene into the adjacent intron. Each round of oligo-cassette mediated PCR walking was performed using multiple restriction enzymes (EcoRI, HindlTJ, Xbal, BglTI, Hinfl). The method can be applied in combination with any restriction endonuclease. Protocol: ds oligo-cassettes were 28 nucleotides long having a 4 nt or 3 (Hinfl) nt overhang. Genomic DNA (250-500 ng) was digested to completion and the restriction enzyme inactivated by heating. Half of the DNA was ligated to 5-50 pmol of oligocassette in a total of 20 y\\. Following dilution to 100 /xl and heat inactivation 1 /d of the ligated product was used for the first round linear PCR with a specific primer which had been biotinylated at its 5'-end. PCR was carried out in 1 xPCR buffer (Cetus), 250 tiM dNTP's, 0.5 /tM specific biotinylated primer and 2.5 units Taq polymerase (Cetus). Since only one primer is present the amplification (50 cycles of 95°C 0.5 mm, 55°C 1 min, 72°C 1 min) is linear and not exponential. Isolation of biotin-labelled products was performed using 25 /xl of washed streptavidin-coated beads (Dynal S.A.). After incubation for 15 min at RT the beads were washed three times with 40 yl of 1 M NaCl in TE buffer followed by three washes with 1 xTE buffer. Supernatants were carefully removed after each washing. Exponential PCR was earned out using the polymer bound DNA as the template as described above except that 35 cycles were carried out and two primers were present, (one complementary to the cassette and the other unbiotinylated but specific to the known sequence). Amplification products were analysed on 1% agarose. After reamplification, specific PCR products were separated on 1 %

175 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023275
2022448
2021172
2020176
2019221
2018220