scholarly journals Comparing the efficacy of three diagnostic tools (microscopic examination, conventional PCR, and Real-Time PCR) in detecting Theileria equi infection among Egyptian equines

2021 ◽  
Author(s):  
Ahmed M. Soliman ◽  
Moaz M. Amer ◽  
Fareed Uddin Memon
Keyword(s):  
Real Time ◽  
Microscopic Examination ◽  
Real Time Pcr ◽  
Protozoan Parasite ◽  
Diagnostic Tools ◽  
Microscopical Examination ◽  
Conventional Pcr ◽  
Theileria Equi ◽  
Diagnostic Assays ◽  
Blood Smears

Abstract Equine theileriosis represents one of the main and serious health problems affecting equines industry globally, that is caused by tick-borne protozoan parasite called T. equi. This study aimed to assess the sensitivity of three diagnostic tools named: microscopic examination of a blood smear, conventional PCR, and Real-Time PCR (qPCR) to detect T. equi among equine population (n = 116) raised in Giza Governorate, Egypt. Microscopic examination of Giemsa-stained blood smears revealed the infection of 16.4% (19/116) of examined equines by T. equi while conventional PCR and qPCR revealed that 29.3% (34/116) and 43.1% (50/116) of examined equines were infected with T. equi respectively. Our results demonstrated that the qPCR had the highest sensitivity (100%) followed by conventional PCR (68%) while microscopic examination had the lowest sensitivity (38%). Furthermore, the negative predictive value (NPV) of qPCR was the highest (100%) compared to conventional PCR and microscopical examination (80.49% and 68.04% respectively) which revealed that all negative cases detected by qPCR were certainly correct compared to the other two diagnostic assays. Therefore, it is highly recommended to incorporate PCR diagnostic assays (conventional PCR and qPCR) alongside microscopic examination to evaluate the epidemiological status of equine theileriosis.

scholarly journals Conventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1757-1762 ◽  
Author(s):  
Ronald J. Sayler ◽  
Courtney Walker ◽  
Fiona Goggin ◽  
Paula Agudelo ◽  
Terrence Kirkpatrick
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Its Region ◽  
Taqman Probe ◽  
Reniform Nematode ◽  
Rotylenchulus Reniformis ◽  
Conventional Pcr ◽  
Diagnostic Assays ◽  
Reniform Nematodes

Reniform nematode (Rotylenchulus reniformis) is a relatively recent introduction into the continental United States that can cause major yield losses on a variety of important crops including cotton and soybeans. DNA sequences from the internal transcribed spacer (ITS) region of this nematode were used to design primers for conventional and real-time PCR, as well as a TaqMan probe. These primers amplified DNA of reniform nematode isolates from a wide geographic range but did not detect genetically related species or other pathogenic nematodes found in production fields including Meloidogyne incognita and Heterodera glycines. Both SYBR green and TaqMan assays reliably quantified as little as 100 fg of reniform nematode DNA, and could be used to quantify as few as five reniform nematodes. An inexpensive and rapid DNA extraction protocol for high throughput diagnostic assays is described.

scholarly journals Fast Identification of Yersinia pestis, Bacillus anthracis and Francisella tularensis Based on Conventional PCR

2013 ◽  
Vol 62 (4) ◽  
pp. 453-455 ◽  
Author(s):  
ALEKSANDRA A. ZASADA ◽  
KAMILA FORMIŃSKA ◽  
KATARZYNA ZACHARCZUK
Keyword(s):  
Public Health ◽  
Real Time ◽  
Francisella Tularensis ◽  
Real Time Pcr ◽  
Health Impact ◽  
Diagnostic Tools ◽  
Conventional Pcr ◽  
The Public ◽  
Detection And Identification ◽  
Appropriate Treatment

Rapid and accurate diagnostic tools for detection and identification of Y pestis, B. anthracis and F. tularensis are essential for timely initial appropriate treatment of exposed individuals, which will be critical to their survival, as well as for reduction of the public health impact and the spread of the disease. The paper presents application of fast polymerases and fast dry electrophoresis in conventional PCR as an alternative for real-time PCR application for detection and identification of the above pathogens. The proposed method takes less than 50 min. to obtain final results of the tests and is cheaper than real-time PCR.

scholarly journals Development of diagnostic assays for rapid and sensitive detection of Phytophthora infecting major spices and plantation crops

1970 ◽  
Vol 27 (2) ◽  
pp. 119
Author(s):  
R T P Pandian, A I Bhat, C N Biju, S Sasi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Black Pepper ◽  
Nuclear Ribosomal Dna ◽  
Diagnostic Tools ◽  
Diagnostic Assays ◽  
Horticultural Crops ◽  
Polymerase Chain ◽  
Plantation Crops ◽  
Epidemiological Significance

Phytophthora, the ubiquitous stramenopile phytopathogen is a major threat to several economically important horticultural crops including spices and plantation crops. Trans-seasonal survival of Phytophthora in plant debris and soil continuum has considerable epidemiological significance as the quiescent propagules often serve as primary foci of infection with inherent potential to trigger epiphytotics in the succeeding season favoured by conducive environmental conditions. Hence, early and rapid detection of over summering propagules is highly imperative to manage Phytophthora induced diseases efficiently and economically. Twelve isolates representing different species of Phytophthora (P. capsici, P. tropicalis, P. palmivora. P. citrophthora and P. meadii) representing hosts such as black pepper, cardamom, nutmeg, coconut, arecanut and cocoa were used to develop nucleic acid-based diagnostic tools viz., polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP) and real-time LAMP. Phytophthora genus-specific primers were designed from the conserved region of nuclear ribosomal DNA. Each of the assays was specific and detected different species of Phytophthora and not other pathogens (Rhizoctonia solani, Pythium vexans, Fusarium oxysporum and Colletotrichum gloeosporioides) and plant samples. Sensitivity assays indicated that, real-time PCR detected Phytophthora upto 1.3 fg, followed by LAMP (13 fg) and PCR (13 pg).

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

Food Control ◽  
2019 ◽  
Vol 98 ◽  
pp. 380-388 ◽  
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

scholarly journals 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 ◽  
2019 ◽  
Vol 103 (2) ◽  
pp. 345-356 ◽  
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.

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

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.

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

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

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

2011 ◽  
Vol 11 (4) ◽  
pp. 418-425 ◽  
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.

scholarly journals Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 152 ◽  
Author(s):  
Vivornpun Sanprasert ◽  
Ruthairat Kerdkaew ◽  
Siriporn Srirungruang ◽  
Sarit Charuchaibovorn ◽  
Kobpat Phadungsaksawasdi ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Simultaneous Detection ◽  
Diagnostic Tools ◽  
Multiple Infections ◽  
Parasitic Infections ◽  
Soil Transmitted Helminths ◽  
Stool Samples

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin–ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

scholarly journals Congenital Malaria in Newborns Delivered to Mothers with Malaria-Infected Placenta in Blue Nile State, Sudan

2020 ◽  
Vol 66 (4) ◽  
pp. 428-434
Author(s):  
Samia A Omer ◽  
Ishag Adam ◽  
Ali Noureldien ◽  
Hadeel Elhaj ◽  
Laura Guerrero-Latorre ◽  
...  
Keyword(s):  
Cord Blood ◽  
Real Time ◽  
Real Time Pcr ◽  
Peripheral Blood ◽  
Molecular Techniques ◽  
Blue Nile ◽  
Blood Smears ◽  
Polymerase Chain ◽  
Thick Smear ◽  
Congenital Malaria

Abstract Diagnosis of congenital malaria is complicated by the low density of the parasite circulating in the cord blood and/or the peripheral blood of the newborns. Molecular techniques are significantly more sensitive than blood smears in detecting low-level parasitemia. This study investigated the prevalence of congenital malaria by the use of the real-time polymerase chain reaction (real-time PCR) in 102 babies born to mothers with microscopically confirmed infected placenta from Blue Nile state, Sudan. At delivery time, placental, maternal peripheral and cord blood samples in addition to samples collected from the newborns’ peripheral blood were examined for malaria infection using Giemsa-stained thick smear and parasite DNA detection by real-time PCR. The overall prevalence of congenital malaria includes the total babies with cord blood parasitaemia and peripheral blood parasitaemia was 18.6 and 56.8% using microscopy and real-time PCR, respectively. Even though all the neonates were aparasitaemic by microscopy, 19 (18.6%) of the babies had congenital malaria detected by real-time PCR, 15 (25.9%) of the babies with congenital malaria were born to mothers with both placental and peripheral blood malaria infections detected using the two techniques. Congenital malaria was significantly associated with cord blood malaria infections, maternal age and maternal haemoglobin level (p < 0.001). This first study investigating congenital malaria in Blue Nile state, Sudan shows that malaria-infected placenta resulted in infant and cord blood infections.

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