Multiplex PCR: Optimization and Application in Diagnostic Virology

Elnifro, E. M.; Ashshi, Ahmad Mohammad; Cooper, R. J.; Klapper, Paul E.

doi:10.1128/cmr.13.4.559-570.2000

Cited by 461 publications

(330 citation statements)

References 71 publications

Supporting

Mentioning

316

Contrasting

Unclassified

Order By: Relevance

“…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%

“…The standard laboratory Detection of ASFV/CSFV by multiplex RT-PCR 553 methods for diagnosis of ASFV and CSFV are mainly based on virus isolation in cell culture, which is tedious and time consuming. The PCR is an alternative rapid detection method of the viruses [6,10]. It has at least the same sensitivity than standard cell cultures and offers much higher sensitivity and specificity than other available rapid methods such as ELISA and immunofluorescence based assays [23,30,35].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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.

View full text Add to dashboard Cite

-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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

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.

View full text Add to dashboard Cite

show abstract

“…However, PCR assays can be time-consuming, laborintensive and expensive, particularly when testing for multiple pathogens in a large number of samples. Multiplex PCR assays are an alternative but their optimization is often difficult [6] and they have only been used for the detection of DNA for a maximum of two tick-borne pathogens [5].…”

Section: Introductionmentioning

confidence: 99%

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Halos

Mavris

Vourc’H³

et al. 2006

Vet. Res.

View full text Add to dashboard Cite

-Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.Ixodes ricinus / Borrelia burgdorferi sensu lato / Anaplasma phagocytophilum / Rickettsia spp. / bacterial diversity

show abstract

“…Thus, the multiplex PCR becomes a highly cost-effective method because of the reduction in labor and reagent and faster detection. [8][9][10] Multiplex PCR has been used for detection of HBV and coinfection with both HAV and HCV. 11 The present study was designed with the aim to develop a rapid, reliable, and cost-effective multiplex PCR assay for the simultaneous detection of both DNA and RNA containing hepatitis viruses which are highly prevalent in India such as HBV, HCV, and HEV.…”

mentioning

confidence: 99%

Multiplex Reverse Transcriptase-PCR for Simultaneous Detection of Hepatitis B, C, and E Virus

Garg

Kumar

Asim

et al. 2016

Journal of Clinical and Experimental Hepatology

View full text Add to dashboard Cite

Introduction: The hepatitis B virus (HBV), HCV, and HEV may occur as singly or concurrently in patients of different kind of liver disease. The rapid, reliable, and cost-effective screening of these pathogens is required for the large epidemiological studies. Therefore, a study has been planned to develop a multiplex Reverse Transcriptase-PCR assay which can be used for the screening of maximum number of pathogens at a time. Methodology: To develop multiplex Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay for simultaneous detection of HBV, HCV, and HEV; the serum samples of 54 patients who were positive either singly or in co-infection with for HBV, HCV, and HEV serologically were screened by uniplex PCR/RT-PCR followed by multiplex RT-PCR for HBV, HCV, and HEV using specific primers. These primers can detect most genotypes of these viruses. Multiplex RT-PCR was done in one tube for the identification of viral DNA/RNA using a mixture of three pairs of specific primers for hepatitis B, C, and E viruses. Representative positive samples of these viruses by uniplex/multiplex RT-PCR were also confirmed by sequencing followed by alignment with reference strains sequence. Results: The specificity of multiplex PCR was 100% with high sensitivity 89%, 87%, and 74% for HBV, HCV, and HEV respectively. The sensitivity and specificity of RT-multiplex PCR demonstrated a good correlation with that of uniplex PCR. Conclusion: The study suggests that multiplex RT-PCR can serve as a simple and reliable assay for rapid, sensitive, and cost-effective method for simultaneous detection of super-infections with HEV particularly in Asian countries as a cause of decompensation of chronic liver disease. ( J CLIN EXP HEPATOL 2016;6:33-39)

show abstract

Multiplex PCR: Optimization and Application in Diagnostic Virology

Cited by 461 publications

References 71 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

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Multiplex Reverse Transcriptase-PCR for Simultaneous Detection of Hepatitis B, C, and E Virus

Contact Info

Product

Resources

About