Comparative assessment of conventional PCR with multiplex real-time PCR using SYBR Green I detection for the molecular diagnosis of imported malaria

For the diagnosis of imported malaria, optical or immunochromatographic methods are known to be less sensitive and less specific than PCR-based methods, which are conversely more complicated and time-consuming. An original strategy, based upon the sequential use of a multiplex competitive real-time PCR detectingPlasmodium falciparumorPlasmodiumspp. infection, followed by, if necessary, a single real-time PCR for species identification, was therefore performed and then tested versus conventional PCR in routine conditions. Conventional PCR has been used since October 1999 in the Department of Parasitology, University Hospitals in Toulouse, as a 2nd line diagnostic method. Out of 183 patients tested, 48 were found to be harbouring a falciparum infection by conventional microscopy, 60 by conventional PCR and 60 by multiplex competitive real-time PCR. Nine further patients had a non-falciparum infection, and concordant species identifications were obtained by both conventional PCR and single real-time PCR. The major value of PCR-based methods, when compared to microscopical techniques, was to ascertain the negativity of a suspect sample. Moreover, real-time PCR allows simplification of the operating procedure, with a diagnosis being made within 2 h.

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A direct comparison of real time PCR on plasma and blood to detect Plasmodium falciparum infection in children

Malaria Journal ◽

10.1186/1475-2875-11-201 ◽

2012 ◽

Vol 11 (1) ◽

Cited By ~ 11

Author(s):

Abigail A Lamikanra ◽

Carlota Dobaño ◽

Alfons Jiménez ◽

Augusto Nhabomba ◽

Hoi P Tsang ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Real Time ◽

Real Time Pcr ◽

Falciparum Infection ◽

Plasmodium Falciparum Infection

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Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

Food Control ◽

10.1016/j.foodcont.2018.11.032 ◽

2019 ◽

Vol 98 ◽

pp. 380-388 ◽

Cited By ~ 3

Author(s):

Xiaofu Wang ◽

Ting Tang ◽

Qingmei Miao ◽

Shilong Xie ◽

Xiaoyun Chen ◽

...

Keyword(s):

Real Time ◽

Transgenic Rice ◽

Real Time Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Processed Foods ◽

Conventional Pcr ◽

Rice Line ◽

Transgenic Rice Line

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A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits

Plant Disease ◽

10.1094/pdis-05-18-0798-re ◽

2019 ◽

Vol 103 (2) ◽

pp. 345-356 ◽

Cited By ~ 4

Author(s):

Yosra Ahmed ◽

Jacqueline Hubert ◽

Céline Fourrier-Jeandel ◽

Megan M. Dewdney ◽

Jaime Aguayo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Culture Media ◽

Direct Detection ◽

Elongation Factor ◽

Single Copy ◽

The United States ◽

Performance Criteria ◽

In Planta ◽

Conventional Pcr

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl−1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl−1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

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Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus

BMC Veterinary Research ◽

10.1186/s12917-020-02399-z ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Xiyu Zhang ◽

Ming Yao ◽

Zhihui Tang ◽

Daning Xu ◽

Yan Luo ◽

...

Keyword(s):

Influenza Virus ◽

Standard Deviation ◽

Real Time ◽

Real Time Pcr ◽

Virus Isolation ◽

Simultaneous Detection ◽

Conventional Pcr ◽

Pcr Assay ◽

Duck Tembusu Virus ◽

Relative Standard

Abstract Background Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. Results The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won’t cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. Conclusion The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

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Establishment of conventional PCR and real-time PCR assays for accurate, rapid and quantitative authentication of four mistletoe species

Phytochemistry ◽

10.1016/j.phytochem.2020.112400 ◽

2020 ◽

Vol 176 ◽

pp. 112400

Author(s):

Wook Jin Kim ◽

Sungyu Yang ◽

Goya Choi ◽

Inkyu Park ◽

Pureum Noh ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Conventional Pcr ◽

Pcr Assays

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Rapid, specific and quantitative polymerase chain reaction (PCR) detection of pathogenic protozoa Entamoeba histolytica for drinking water supply

Water Science & Technology Water Supply ◽

10.2166/ws.2011.057 ◽

2011 ◽

Vol 11 (4) ◽

pp. 418-425 ◽

Cited By ~ 1

Author(s):

S. W. Lam ◽

H. B. Zhang ◽

L. Yu ◽

C. H. Woo ◽

K. N. Tiew ◽

...

Keyword(s):

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Genomic Dna ◽

Tap Water ◽

Pcr Detection ◽

Raw Water ◽

Conventional Pcr ◽

Polymerase Chain ◽

Species Specific

In this study, a quantitative species-specific polymerase chain reaction (PCR) method to rapidly detect E. histolytica in water is developed. First, the specificity of E. histolytica PCR detection was verified by using species-specific primers of 16S-like rRNA genes to clearly differentiate it from the closely related amoebae species E. dispar and E. moshkovskii. The sensitivity of this method was subsequently determined using purified E. histolytica genomic DNA and culture cells as PCR reaction templates. Results indicated that conventional PCR visualized on 1% agarose gel was able to detect as low as 0.02 pg genomic DNA and 5 cells, while real-time PCR could detect 0.01 pg genomic DNA and 2 cells of E. histolytica. The protocols for E. histolytica PCR detection in real water samples were then optimized by spiking E. histolytica cells into tap water and reservoir raw water samples. A two-round centrifugation treatment to concentrate amoeba cells directly as a PCR template was the most effective way to detect E. histolytica in spiked tap water samples, while DNA extraction after concentrating amoeba cells was required for spiked reservoir raw water samples. The detection limit of 50 E. histolytica cells in 100 ml tap water was achieved in 2 h from sample collection to real-time PCR data readout. With these established protocols, 78 tap water samples, 11 reservoir raw water samples and 4 feed water samples from Singapore water supply systems were analyzed by both conventional PCR and real-time PCR methods. No E. histolytica cell was detected in tested samples.

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Impact of Routine Real-Time PCR Testing of Imported Malaria over 4 Years of Implementation in a Clinical Laboratory

Journal of Clinical Microbiology ◽

10.1128/jcm.00195-13 ◽

2013 ◽

Vol 51 (6) ◽

pp. 1850-1854 ◽

Cited By ~ 7

Author(s):

S. Shokoples ◽

S. N. Mukhi ◽

A. N. Scott ◽

S. K. Yanow

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Laboratory ◽

Imported Malaria

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DNA recovery from archived RDTs for genetic characterization of Plasmodium falciparum in a routine setting in Lambaréné, Gabon

Malaria Journal ◽

10.1186/s12936-019-2972-y ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 5

Author(s):

The Trong Nguyen ◽

Brice Nzigou Mombo ◽

Albert Lalremruata ◽

Erik Koehne ◽

Rella Zoleko Manego ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Real Time ◽

Real Time Pcr ◽

Rural Areas ◽

Large Scale ◽

Genetic Material ◽

Chloroquine Resistance ◽

Dna Recovery ◽

Parasite Populations ◽

Pcr Targeting

Abstract Background Rapid diagnostic tests (RDTs) have been described as a source of genetic material to analyse malaria parasites in proof-of-concept studies. The increasing use of RDTs (e.g., in focal or mass screening and treatment campaigns) makes this approach particularly attractive for large-scale investigations of parasite populations. In this study, the complexity of Plasmodium falciparum infections, parasite load and chloroquine resistance transporter gene mutations were investigated in DNA samples extracted from positive RDTs, obtained in a routine setting and archived at ambient temperature. Methods A total of 669 archived RDTs collected from malaria cases in urban, semi-urban and rural areas of central Gabon were used for P. falciparum DNA extraction. Performance of RDTs as a source of DNA for PCR was determined using: (i) amplification of a single copy merozoite surface protein 1 (msp1) gene followed by highly sensitive and automated capillary electrophoresis; (ii) genotyping of the pfcrt gene locus 72–76 using haplotype-specific-probe-based real-time PCR to characterize chloroquine resistance; and, (iii) real-time PCR targeting 18S genes to detect and quantify Plasmodium parasites. Results Out of the 669 archived RDTs, amplification of P. falciparum nucleic materials had a success rate of 97% for 18S real-time PCR, and 88% for the msp1 gene. The multiplicity of infections (MOI) of the whole population was 2.6 (95% CI 2.5–2.8). The highest number of alleles detected in one infection was 11. The MOI decreased with increasing age (β = − 0.0046, p = 0.02) and residence in Lambaréné was associated with smaller MOIs (p < 0.001). The overall prevalence of mutations associated with chloroquine resistance was 78.5% and was not associated with age. In Lambaréné, prevalence of chloroquine resistance was lower compared to rural Moyen-Ogooué (β = − 0.809, p-value = 0.011). Conclusion RDT is a reliable source of DNA for P. falciparum detection and genotyping assays. Furthermore, the increasing use of RDTs allows them to be an alternative source of DNA for large-scale genetic epidemiological studies. Parasite populations in the study area are highly diverse and prevalence of chloroquine-resistant P. falciparum remains high, especially in rural areas.

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