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 PO 8441 EXPERIMENTAL COMPARISON OF SENSITIVITY OF LAMP AND REAL-TIME PCR

2019 ◽  
Vol 4 (Suppl 3) ◽  
pp. A37.3-A38
Author(s):  
Jacques Kaboré ◽  
Hamidou Ilboudo ◽  
Charlie FA Compaoré ◽  
Oumou Camara ◽  
Mohamed Bamba ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Filter Paper ◽  
Real Time Pcr ◽  
Sleeping Sickness ◽  
Taqman Probe ◽  
Experimental Comparison ◽  
Analytical Sensitivity ◽  
Molecular Tools ◽  
Detection Techniques

BackgroundHuman African trypanosomiasis, or sleeping sickness, remains a serious problem in tropical Africa. Timely diagnosis of this disease requires systematic population screening, particularly for Trypanosoma brucei gambiense, which has a long asymptomatic period.The lack of sensitivity and specificity of conventional diagnostic tests has led in recent years to the use of molecular tools. Amplification of parasite-specific DNA sequences significantly improved diagnosis of infection. However, these molecular tools still have some limitations especially in the case of low parasitaemia. Furthermore, research is still needed to make molecular detection a real control tool for the fight against sleeping sickness. The purpose of this study is to determine the threshold of sensitivity of real-time PCR using the 18S and TgsGp primers and of the LAMP technique, applied in the DiTECT-HAT project as molecular reference tests.MethodsWe used serial dilutions containing 0, 1, 10, 100, 103, 104, 105, 106 parasites per ml of blood. Samples were extracted, and DNA was amplified.ResultsThe analytical sensitivity of the 18S real-time PCR with the Taqman probe of the filter paper samples is 100 parasites/ml and that of the TgsGp real-time PCR with the Taqman probe of filter paper samples is 104 parasites/ml. For Lamp technique, the analytical sensitivity is 103 parasites/ml.ConclusionThis study shows that a ‘negative PCR’ would not mean ‘no parasite’. It suggests that DNA detection techniques should still be improved.

scholarly journals Development of a Real-Time PCR based protocol for quantifying Radopholus similis in field samples

1970 ◽  
pp. 52-60
Author(s):  
P B Krishna, S J Eapen
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Its Region ◽  
Pcr Primers ◽  
Soil Samples ◽  
Radopholus Similis ◽  
Efficient Tool ◽  
Field Samples ◽  
Burrowing Nematode

The burrowing nematode, Radopholus similis, is an obligate migratory endoparasite. Currently detection of this nematode is carried out mostly by physically extracting them from soil and then observing under a light microscope. To identify this nematode, a thorough knowledge about their morphological features is quite indispensable. Developing a DNA based detection technique makes it more convenient and accurate in detection. Though PCR based methods have been reported by earlier workers, developing a Real-Time PCR based method will enable estimating their population in field samples. In this study, Real-Time PCR primers were designed using the DNA sequences from the ITS region of R. similis. It can detect R. similis up to the limit of 100 fg μL-1 DNA. The real time PCR based detection serves as an efficient tool for the detection and estimation of this nematode from soil samples.

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.

Patient Specific LymphoTrack™ Assay: Design and Validation of a Robust Real Time PCR Based Igh MRD Assay

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4876-4876
Author(s):  
Ninad D Pendse ◽  
Alisa Ching ◽  
Jeffrey Miller
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Taqman Probe ◽  
Patient Specific ◽  
Specific Primer ◽  
Specificity And Sensitivity ◽  
Allele Specific ◽  
Primer Sets

Abstract Introduction: Chronic lymphocytic leukemia is a clonal proliferation and accumulation of neoplastic small B cells in the peripheral blood, bone marrow and lymph nodes. Many patients with CLL relapse even after aggressive therapies. Several studies have suggested that residual leukemic cells are responsible for relapse. Hence, it is important to have a highly sensitive assay to detect Minimal Residual Disease (MRD) in patients during treatment or when the patient is in morphologic remission. Assays that test for patient specific immunoglobulin heavy chain (IGH) gene rearrangement should permit higher sensitivity for MRD detection. We developed a real-time PCR based MRD assay (LymphoTrack™ assay) that has a minimum limit of detection of one tumor cell in 10,000 normal cells, which is more sensitive than FACS based methods. Methods: Clonal, patient-specific IGH DNA sequences were obtained from 7 patient samples. Multiple DNA samples from normal human tonsils were used as normal polyclonal controls for all studies. Primer sets were designed using an upstream patient-specific primer paired with either a reverse allele-specific primer targeting the JH intron or a second patient-specific primer. In either of the methods, the primer pair was combined with an allele specific TaqMan probe targeted at an appropriate region of the IGH sequence. For any given patient sequence, multiple primer sets were designed, and then run on a series of specificity and sensitivity tests to select the best primer set. First to test for specificity, all primer sets were run with various lots of normal tonsil DNA and water using SYBR green detection. The primer sets that didn’t amplify tonsil DNA or had an amplification product with a Ct > 40 were further tested for specificity with the same tonsil DNAs and water by including the appropriate TaqMan probe for that patient. The primer/probe sets that did not amplify either tonsil or water were then tested with the patient DNA. Diagnostic patient DNA was serially diluted into tonsil DNA to verify the ability of the primer/probe set to identify at least a 10−4 dilution of the patient DNA. All testing was done in triplicate to determine intra-assay concordance. Lastly, testing was repeated to verify inter-assay repeatability. Results: 5–7 sets of primers were designed for each patient sample. Real time PCR data were used to select an optimal primer set for each patient. Optimal primer sets, selected on the basis of specificity, were then tested to determine sensitivity. In 6 of 7 patients the optimal primer set achieved a sensitivity of 10−4 or higher. In 5 of the 7 cases test of tonsil never generated a signal that reached threshold even at 50 cycles. In the test for other two patients, tonsil was amplified at Ct > 45. Tests from 5 of the 7 sets did not amplify product from cell lines with known IGH gene rearrangements indicating the specificity of the primer set. 2 sets amplified DNA from 1 cell line each at a very high Ct value. Conclusion: We designed and developed 7 patient specific MRD tests in times averaging 3–4 weeks. In this study, we were able to design a primer set with a sensitivity of at least 10−4 in 6 of the 7 cases without compromising the specificity. Our experience with this patient set suggests that real time PCR based MRD tests can be developed quickly and efficiently using our methodology to provide assays of both high sensitivity and specificity.

Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

2005 ◽  
Vol 68 (6) ◽  
pp. 1217-1221 ◽  
Author(s):  
PAVEL KRCMAR ◽  
EVA RENCOVA
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Single Species ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Vertebrate Species ◽  
Target Species ◽  
Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

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 Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

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.

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