Development and validation of real-time PCR assays based on novel molecular markers for the simultaneous detection and identification of Globodera pallida, G. rostochiensis and Heterodera schachtii

Nematology ◽  
2017 ◽  
Vol 19 (7) ◽  
pp. 789-804 ◽  
Author(s):  
Sylvie Gamel ◽  
Aude Letort ◽  
Didier Fouville ◽  
Laurent Folcher ◽  
Eric Grenier
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Nematode Species ◽  
Globodera Pallida ◽  
Heterodera Schachtii ◽  
Cyst Nematodes ◽  
Detection And Identification ◽  
Template Dna

Considering the growing trade of seed potato, reliable diagnostic protocols are required for the detection of regulated nematode species. In this study, a specific and sensitive multiplex Taqman-based real-time PCR method was developed in order to detect and identifyGlobodera pallida,G. rostochiensisandHeterodera schachtii. The newly designed primers and probes enabled the detection of all the target populations tested and with no cross-reaction for closely related non-target species (55 populations tested). The limit of detection (LOD) was one juvenile forG. rostochiensisandG. pallidaand five juveniles forH. schachtii. For monitoring potato cyst nematodes, this analytical tool would extend the number of cyst investigated as five juveniles can be detected among 50 cysts in a sample. Furthermore, this multiplex assay detects DNA of the three targeted species in template DNA obtained directly from float material after nematode extraction from soil.

DEVELOPMENT OF SYBR GREEN-BASED REAL-TIME PCR FOR THE DETECTION OF CANINE, FELINE AND PORCINE PARVOVIRUSES

2014 ◽  
Vol 40 (01) ◽  
pp. 1-9 ◽  
Author(s):  
Chao-Nan Lin ◽  
Chi-Hsien Chien ◽  
Ming-Tang Chiou ◽  
Jou-Wei Wang ◽  
Ya-Ling Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Sybr Green ◽  
Porcine Parvovirus ◽  
Diagnostic Laboratory ◽  
Vp2 Gene ◽  
Coefficients Of Variation ◽  
Causes Of Mortality

Parvovirus is now considered to be one of the most important infectious agents affecting canine, feline and porcine domestic animals. Canine parvovirus 2 (CPV 2) and feline parvovirus (FPV) are major infectious causes of mortality in puppies and kittens. Both CPV 2 and FPV have recently been shown to infect both dogs and cats. The characteristic symptoms of canine, feline and porcine parvovirus disease are intestinal hemorrhage with severe bloody diarrhea, leukopenia and reproductive failure, respectively. In this work, we describe the development of a novel real-time PCR system that is based on the use of SYBR Green and that allows the simultaneous detection of VP2 gene of CPV, FPV and porcine parvovirus (PPV). This system yielded low coefficients of variation for intra-assay and inter-assay variabilities. This novel SYBR Green-based real-time PCR assay was sensitive, specific and reliable for the amplification of CPV 2, FPV and PPV DNA, with a reproducible limit of detection of as few as 10 copies/μL of target DNA per reaction. The methods described in this study have been used successfully in our veterinary diagnostic laboratory and have been shown to be helpful tools for the diagnosis and quantification of parvovirus infection in canines, felines and swine.

scholarly journals Comparative genomics among three cyst nematode species reveals distinct evolutionary histories among effector families and an irregular distribution of effector-associated promoter motifs

2021 ◽  
Author(s):  
Joris J.M. van Steenbrugge ◽  
Sven van den Elsen ◽  
Martijn Holterman ◽  
Jose L. Lozano-Torres ◽  
Vera Putker ◽  
...  
Keyword(s):  
Host Plant ◽  
Reference Genome ◽  
Nematode Species ◽  
Cyst Nematode ◽  
Globodera Pallida ◽  
Heterodera Schachtii ◽  
Cyst Nematodes ◽  
Promoter Regions ◽  
Long Read ◽  
Additional Reference

Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype-specific host plant resistances are an essential handle for PCN control. However, the poor delineation of G. pallida pathotypes hampers the efficient use of available host plant resistances. Long-read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCN after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression (DOG boxes) were present in all three cyst nematode species, their presence is not a necessity for dorsal gland-produced effectors. Notably, DOG box dosage was only loosely correlated with expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Re-sequencing of PCN populations with deviant virulence characteristics will allow for the linking of these characteristics with the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion.

scholarly journals Multiplex Real-Time PCR Assays for the Identification of the Potato Cyst and Tobacco Cyst Nematodes

Plant Disease ◽  
2010 ◽  
Vol 94 (8) ◽  
pp. 959-965 ◽  
Author(s):  
Mark K. Nakhla ◽  
Kristina J. Owens ◽  
Wenbin Li ◽  
Gang Wei ◽  
Andrea M. Skantar ◽  
...  
Keyword(s):  
Real Time ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Globodera Pallida ◽  
Cyst Nematodes ◽  
Detection And Identification ◽  
Polymerase Chain ◽  
Probe Set ◽  
Internal Transcribed Spacer Rdna ◽  
Quantitative Pcr Assay

TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCNs) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time polymerase chain reaction (PCR). One tube contained a primer-probe set specific for G. pallida (pale potato cyst nematode) multiplexed with another primer-probe set specific for G. rostochiensis (golden potato cyst nematode). A second tube consisted of the G. pallida-specific primer-probe set multiplexed with a primer-probe set specific for G. tabacum (the morphologically similar tobacco cyst nematode). This internal transcribed spacer rDNA-based system was specific for the Globodera spp. of interest and successfully identified several populations of PCN. This rapid, sensitive, and specific quantitative PCR assay presents a useful tool for PCN regulatory response and management programs.

Conventional and real-time PCR-based species identification and diversity of potato cyst nematodes (Globodera spp.) from Victoria, Australia

Nematology ◽  
2008 ◽  
Vol 10 (4) ◽  
pp. 471-478 ◽  
Author(s):  
Lila Nambiar ◽  
James Cunnington ◽  
Motiul Quader
Keyword(s):  
Sequence Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Binding Sites ◽  
Sequence Variation ◽  
Control Strategies ◽  
Globodera Pallida ◽  
Pcr Analysis ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes

AbstractPCR (conventional and real-time) and DNA sequence analysis were used to identify species and genotypes of potato cyst nematodes from sites in Victoria, Australia. Only Globodera rostochiensis was detected. Sequence analyses of these isolates of PCN have confirmed the PCR-based results and have revealed the presence of genetically diverse populations in infested fields. However, the sequence variation was not in the diagnostic primer binding sites. The melting peaks, from multiplex real-time PCR analysis, for Globodera pallida and G. rostochiensis were 83.3 and 88.7, respectively. The importance of DNA extraction, PCR and sequence analysis for the molecular identification of PCN is discussed. This study has significant implications for detecting species of PCN in order to monitor/develop control strategies for the PCN of quarantine importance.

scholarly journals Development of Real-Time and Conventional PCR Assays for Identifying Stubby Root Nematode Paratrichodorus allius

Plant Disease ◽  
2017 ◽  
Vol 101 (6) ◽  
pp. 964-972 ◽  
Author(s):  
Danqiong Huang ◽  
Guiping Yan ◽  
Andrea M. Skantar
Keyword(s):  
Real Time ◽  
Molecular Diagnosis ◽  
Real Time Pcr ◽  
Tobacco Rattle Virus ◽  
Nematode Species ◽  
Detection Sensitivity ◽  
Conventional Pcr ◽  
Nematode Communities ◽  
Detection And Identification ◽  
Pcr Assays

Paratrichodorus allius is an important pest on many crops, particularly on potato due to its ability to transmit Tobacco rattle virus causing corky ringspot disease on tubers. Detection and identification of P. allius are important for effective disease management. In this study, a rapid and reliable molecular diagnosis of this nematode targeting internal transcribed spacer ribosomal DNA was established. The specificity of the designed primers was evaluated using 29 nematode species and results showed that a single amplicon was produced from DNA of P. allius only. Detection sensitivity analysis indicated that a 9.6 × 10−4 ng of DNA template could be detected by conventional PCR and 1.92 × 10−4 ng of DNA by real-time PCR. The PCR assays amplified DNA of stubby root nematodes isolated from 18 soil samples in North Dakota and Minnesota, which were confirmed as P. allius by sequencing. Both conventional PCR and real-time PCR assays amplified target nematodes from complex nematode communities, supporting the success of this molecular diagnosis of P. allius. This is the first report of P. allius identification using the real-time PCR method and from nematode communities with other nematodes using conventional PCR. The new PCR assays provide rapid species identification and are suitable for use in diagnostic laboratories and detection of field infestations with P. allius.

scholarly journals Development of Simple Multiplex Real-Time PCR Assays for Foodborne Pathogens Detection and Identification On Lightcycler

2017 ◽  
Vol 40 (1) ◽  
pp. 53-58 ◽  
Author(s):  
Avo Karus ◽  
Fabrizio Ceciliani ◽  
Armand Sanches Bonastre ◽  
Virge Karus
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Melting Curve ◽  
Food Contamination ◽  
Shigella Boydii ◽  
Food Borne Pathogens ◽  
Detection And Identification ◽  
Food Borne

Abstract Most acute intestinal diseases are caused by food-borne pathogens. A fast and simple real-time PCR-based procedure for simultaneous detection of food contamination by any of the five food-borne pathogens: Campylobacter jejuni, Mycobacterium bovis, Enterobacter sakazaki, Shigella boydii, Clostridium perfrigens using multiplex EvaGreen real-time PCR for LightCycler was developed and evaluated. Real-time qPCR showed excellent sensitivity. Tm calling and Melting Curve Genotyping (MCG) were used for analysis of PCR product melting curves. The Melting Curve Genotyping option showed good performance for discrimination of positive samples containing DNA of single pathogen or pathogen mixtures from negative samples.

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