Multiplex PCR: advantages, development, and applications.

Edwards, Michael C.; Gibbs, Richard A.

doi:10.1101/gr.3.4.s65

Cited by 341 publications

(219 citation statements)

References 59 publications

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“…The most common problem is that quite a number of primers have to be used in the same reaction tube or well and these molecules may interact with each other, which may block the reaction [7,9,10,14,22]. A further problem may be the reliable identification of the various PCR products.…”

Section: Discussionmentioning

confidence: 99%

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

Agüero

Fernández²,

Romero

et al. 2004

Vet. Res.

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-The development and standardisation of a novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay is presented for the simultaneous and differential diagnosis of African swine fever (ASF) and Classical swine fever (CSF). The method uses two primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR products. Universal detection of ASF and CSF virus strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the two viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues. Additional confirmatory tests of the ASF and CSF virus amplicon specificity, based on restriction endonuclease analysis with BsmA I or Ban II, respectively, are also described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of both diseases, even before the appearance of the first clinical signs. A study of 150 positive field samples from several ASF and CSF outbreaks showed the suitability of this method for a rapid (less than five hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for CSFV was also developed and standardised as a powerful tool for fast and early diagnosis of the disease.

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Section: Discussionmentioning

confidence: 99%

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

Agüero

Fernández²,

Romero

et al. 2004

Vet. Res.

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“…Furthermore, multiplex PCR is able to detect multiple target DNA sequences in a single reaction, with the simultaneous identification of two or more kinds of pathogens, simplifying the workflow and processing time, which are significantly reduced (Bilgic et al 2013), among other advantages (Edwards and Gibbs 1994).…”

Section: Discussionmentioning

confidence: 99%

Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honey bee products

Guimarães-Cestaro¹,

Serrão²,

Message³

et al. 2016

JHR

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Honey bees are responsible for pollinating many native and cultivated plant species. These insects can be affected by many pathogens, including fungi and bacteria, both of which can form spores that are easily dispersed within the colony by means of the stored products, among other routes. The objective of this study was to develop a method to detect spores of the honey bee pathogens Nosema apis, Nosema ceranae, Ascosphaera apis and Paenibacillus larvae in samples of honey, bee pollen and royal jelly. The method was standardized for each product individually, and then analyzed by monoplex and multiplex PCR, which showed the same detection thresholds: 1.25 spores/mL of honey for N. ceranae; 7.5 spores/mL of honey for A. apis; and 0.4 spore/mL of honey for P. larvae, respectively. The standardized technique was effective and rapid for the detection of these pathogens in bee products and can be used for the establishment of official methods of sanitary control of bee products, considering the growing national and international trade of these products and the movement or migration of colonies between regions.

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“…However, the development of a multiplex qPCR using the same gene is difficult due to the competition for resources. Moreover, when one target is more abundant than another, unequal amplification will occur and the differences will be enhanced during each cycle, leading to artifact and misinterpretation of the data (Edwards and Gibbs, 1994). Furthermore, the use of the 16S rDNA gene, even though it is a highly homologous target sequence for practically all bacteria which can be used for detection (Barken et al, 2007;Mothershed and Whitney, 2006), can deliver some technical problems.…”

Section: Comparison Between Taqman Sybr Green and Microscopymentioning

confidence: 99%

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

Vanysacker

Denis

Roels

et al. 2014

Journal of Microbiological Methods

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Candidatus Microtrhix parvicella is one of the most common filamentous bacteria reported to be involved in bulking and foaming problems in activated sludge plants worldwide. In order to detect and quantify both M. parvicella and Microthrix calida by quantitative PCR (qPCR), primers targeting 16S rDNA genes were designed. The qPCR reaction was optimized by using the TaqMan technology and an internal positive control was included to ensure the absence of PCR inhibitors. A total of 29 samples originating from different wastewater treatment plants were analyzed and the results were compared by using conventional microscopy, fluorescent in situ hybridization and an existing SYBR Green-based assay. Our assay showed a 100% specificity for both M. parvicella and M. calida, a sensitivity of 2.93 × 10 9 to 29 copy numbers/reaction, an amplification efficiency of 93% and no PCR inhibition. By performing a spiking experiment including different Microthrix concentrations, recovery rates ranging from 65 to 98% were obtained. A positive correlation with the SYBR Green assay (R 2 = 0.85) was found and most of the samples were in accordance with the microscopical observation. In comparison with SYBR Green assay, the probe-based TaqMan assay had a much lower detection limit. Compared with microscopy, some samples had a lower or higher enumeration when using qPCR. In conclusion, a qPCR method is forwarded here that could be useful as an early warning tool for fast and reliable detection of Microthrix in for instance sludge bulking events.

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Multiplex PCR: advantages, development, and applications.

Cited by 341 publications

References 59 publications

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honey bee products

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

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