Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

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The epidemiology of amoebiasis in Thi-Qar province, Iraq (2015-2020): Differentiation of Entamoeba histolytica/Entamoeba dispar using nested PCR and real-time PCR

Epidemiology and Health ◽

10.4178/epih.e2021034 ◽

2021 ◽

pp. e2021034

Author(s):

Mohammed Hassan Flaih ◽

Ruaa M. Khazaal ◽

Manar K. Kadhim ◽

Khwam R. Hussien ◽

Falah A. B. Alhamadani

Keyword(s):

Real Time ◽

Entamoeba Histolytica ◽

Real Time Pcr ◽

Nested Pcr ◽

Entamoeba Dispar

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Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Memórias do Instituto Oswaldo Cruz ◽

10.1590/s0074-02762011000600008 ◽

2011 ◽

Vol 106 (6) ◽

pp. 691-700 ◽

Cited By ~ 20

Author(s):

Giselle FMC Lima ◽

José E Levi ◽

Marcelo P Geraldi ◽

Maria Carmen A Sanchez ◽

Aluísio AC Segurado ◽

...

Keyword(s):

Comparative Analysis ◽

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

Large Scale ◽

Malaria Diagnosis ◽

Serological Diagnosis ◽

Blood Samples

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Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽

10.2478/s11536-007-0029-z ◽

2007 ◽

Vol 2 (3) ◽

pp. 271-279 ◽

Cited By ~ 1

Author(s):

Koray Ergunay ◽

Gulcin Altinok ◽

Bora Gurel ◽

Ahmet Pinar ◽

Arzu Sungur ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

San Francisco ◽

Real Time Pcr ◽

Nested Pcr ◽

Parvovirus B19 ◽

Etiologic Agent ◽

Hydrops Fetalis ◽

Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

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Simultaneous Detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in Fecal Samples by Using Multiplex Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.42.3.1220-1223.2004 ◽

2004 ◽

Vol 42 (3) ◽

pp. 1220-1223 ◽

Cited By ~ 255

Author(s):

J. J. Verweij ◽

R. A. Blange ◽

K. Templeton ◽

J. Schinkel ◽

E. A. T. Brienen ◽

...

Keyword(s):

Real Time ◽

Entamoeba Histolytica ◽

Cryptosporidium Parvum ◽

Real Time Pcr ◽

Giardia Lamblia ◽

Simultaneous Detection ◽

Fecal Samples

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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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Detection and Quantification of Human Adenoviruses in Surface Waters by Nested PCR, TaqMan Real-Time PCR and Cell Culture Assays

Water Air & Soil Pollution ◽

10.1007/s11270-007-9608-5 ◽

2007 ◽

Vol 191 (1-4) ◽

pp. 83-93 ◽

Cited By ~ 27

Author(s):

M. Muscillo ◽

M. Pourshaban ◽

M. Iaconelli ◽

S. Fontana ◽

A. Di Grazia ◽

...

Keyword(s):

Cell Culture ◽

Real Time ◽

Surface Waters ◽

Real Time Pcr ◽

Nested Pcr ◽

Human Adenoviruses ◽

Detection And Quantification

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Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

Scientific Reports ◽

10.1038/s41598-021-00968-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tsui-Kang Hsu ◽

Jung-Sheng Chen ◽

Hsin-Chi Tsai ◽

Chi-Wei Tao ◽

Yu-Yin Yang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

In Silico ◽

Environmental Samples ◽

Method Development ◽

Small Volume ◽

Detection Efficiency ◽

Genotyping Method ◽

Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

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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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