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.

Quantification of viable eggs of the potato cyst nematodes (Globodera spp.) using either trehalose or RNA-specific Real-Time PCR

Nematology ◽  
2014 ◽  
Vol 16 (10) ◽  
pp. 1219-1232 ◽  
Author(s):  
Johanna E. Beniers ◽  
Thomas H. Been ◽  
Odette Mendes ◽  
Marga P.E. van Gent-Pelzer ◽  
Theo A.J. van der Lee
Keyword(s):  
Real Time ◽  
Visual Inspection ◽  
Membrane Integrity ◽  
Globodera Pallida ◽  
Potato Cyst Nematodes ◽  
Original Sample ◽  
Rt Pcr ◽  
Cyst Nematodes ◽  
Pcr Assay ◽  
Field Samples

Two novel methods for the quantitative estimation of the number of viable eggs of the potato cyst nematodes (Globodera pallida and G. rostochiensis) were tested and compared with visual inspection. One is based on the loss of membrane integrity upon death and uses trehalose (a disaccharide) as a marker, the second test exploits the rapid degeneration of mRNA upon decease with a RNA-specific Real-Time Polymerase Chain Reaction (RT-PCR) assay. The viability of eggs in suspensions with different numbers of eggs was determined morphologically and was compared with both trehalose and elongation-factor-1-alpha (EF1α) mRNA measurements. The trehalose assay provided results that were close to those of the visual assessment using a microscope but only when samples contained low numbers of eggs. The lowest detectable value is 1.1 egg in the original sample and small differences in the number of viable eggs can be detected. Unfortunately, trehalose measurements reached a saturation limit at 1 cyst 10 μl−1; therefore, samples with nematode numbers above 262 eggs have to be diluted. The presence of dead cysts did not have a negative effect on the trehalose measurements. However, the use of egg suspensions instead of encysted eggs improved both the trehalose absorbance and the reliability of the measurements. When cysts were exposed to a treatment with allylisothiocyanate, the trehalose measurement detected the presence of more viable eggs than a hatching assay. The RT-PCR assay required a minimum of 30 eggs before detection occurred but can detect up to 8000 eggs in a 25 μl sample, which is an advantage when samples with high PCN infestations have to be processed. However, the confidence intervals (CI) of the RT-PCR assay are larger than those of the trehalose assay, which results in a high variation of single measurements. For example, at a density of 210 eggs in the original sample the 95% CI for the trehalose assay covers 191-228 eggs, and the 95% CI for the RT-PCR assay for G. pallida lies between 73 and 602 eggs and for G. rostochiensis between 59 and 745 eggs. Trials with field samples using both methods supported the laboratory tests. 95% of the field samples tested with the trehalose assay lie within the CI of the standard curve compared to 58% of the RT-PCR tested samples for G. pallida. The measurements of the field samples of G. pallida and G. rostochiensis populations using both methods resulted in larger numbers of viable eggs being detected compared to a hatching test. Neither of the investigated methods in their current state of development is optimal for use as a substitute for the visual inspection used in monitoring labs. The variance of the RT-PCR assay is too high if used for quantitative monitoring; the density range of eggs that can be detected using the trehalose assay is too small.

Failure to detect capsule gene bexA in Haemophilus influenzae types e and f by real-time PCR due to sequence variation within probe binding sites

2005 ◽  
Vol 54 (5) ◽  
pp. 453-455 ◽  
Author(s):  
I.-C Sam ◽  
M Smith
Keyword(s):  
Haemophilus Influenzae ◽  
Real Time ◽  
Real Time Pcr ◽  
Binding Sites ◽  
Sequence Variation ◽  
Pcr Assay ◽  
Do So

Detection of the conserved capsule gene bexA is used to distinguish capsulate from non-capsulate Haemophilus influenzae. While developing a real-time PCR assay to detect bexA, it was found that bexA probes produced a detectable signal for H. influenzae types a to d, but failed to do so for H. influenzae types e and f. Sequencing revealed differences compared with H. influenzae types a to d within probe binding sites. To prevent misclassification of strains as non-capsulate, assays must detect all capsular types.

scholarly journals Detection and Quantification of Plectosphaerella cucumerina, a Potential Biological Control Agent of Potato Cyst Nematodes, by Using Conventional PCR, Real-Time PCR, Selective Media, and Baiting

2003 ◽  
Vol 69 (8) ◽  
pp. 4788-4793 ◽  
Author(s):  
S. D. Atkins ◽  
I. M. Clark ◽  
D. Sosnowska ◽  
P. R. Hirsch ◽  
B. R. Kerry
Keyword(s):  
Biological Control ◽  
Population Size ◽  
Real Time ◽  
Real Time Pcr ◽  
Biological Control Agent ◽  
Control Agent ◽  
Taqman Probe ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes ◽  
Plectosphaerella Cucumerina

ABSTRACT Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

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.

scholarly journals Taxonomy, Morphological and Molecular Identification of the Potato Cyst Nematodes, Globodera pallida and G. rostochiensis

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 184
Author(s):  
John Wainer ◽  
Quang Dinh
Keyword(s):  
Life Cycle ◽  
Molecular Identification ◽  
Soil Sampling ◽  
Globodera Pallida ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes ◽  
Primary Focus

The scope of this paper is limited to the taxonomy, detection, and reliable morphological and molecular identification of the potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis. It describes the nomenclature, hosts, life cycle, pathotypes, and symptoms of the two species. It also provides detailed instructions for soil sampling and extraction of cysts from soil. The primary focus of the paper is the presentation of accurate and effective methods to identify the two principal PCN species.

ID: 316 Real-time PCR analysis of BCL2L12, a novel member of BCL2 family of the apoptosis-related genes, in human leukemia.

2006 ◽  
Vol 4 (s1) ◽  
pp. 82-82
Author(s):  
K. Floros ◽  
H. Thomadaki ◽  
S. Pavlovic ◽  
M. Talieri ◽  
M. Colovic ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Human Leukemia ◽  
Bcl2 Family

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Berta Fidalgo ◽  
Elisa Rubio ◽  
Victor Pastor ◽  
Marta Parera ◽  
Clara Ballesté-Delpierre ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Enteric Pathogens ◽  
Pcr Analysis ◽  
Culture Methods ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Link Type ◽  
Microbiological Culture ◽  
Micro Organisms

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

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