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The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei
- Source :
- Nematology. 16:789-796
- Publication Year :
- 2014
- Publisher :
- Brill, 2014.
-
Abstract
- A real-time quantitative PCR assay was developed for the accurate detection and quantification of the root-lesion nematode, Pratylenchus thornei. A qPCR primer set, including two primers and a probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimised by using the primers with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 62 to 69°C. Based on the Ct values, we retained the program with an annealing temperature of 69°C. The specificity of the qPCR assay including the probe was confirmed by the lack of amplification of DNA from 47 populations belonging to 15 other Pratylenchus species and nine isolates from P. thornei. The assay was very sensitive as it was able to detect a single individual of P. thornei, even when mixed with up to 80 individuals of P. penetrans. DNA was extracted from exactly 80 P. thornei individuals. A dilution series from this DNA resulted in a standard curve showing a highly significant linearity between the Ct values and the dilution rates (; slope = −3.38; ). The qPCR assay developed in this study proved to be specific and sensitive, thus providing a fast and accurate tool for detection and quantification of this pathogen during research, as well as for diagnostic labs.
- Subjects :
- biology
biology.organism_classification
Molecular biology
Standard curve
chemistry.chemical_compound
Real-time polymerase chain reaction
chemistry
SYBR Green I
Primer (molecular biology)
Pratylenchus thornei
Pratylenchus
Agronomy and Crop Science
Gene
Ecology, Evolution, Behavior and Systematics
DNA
Subjects
Details
- ISSN :
- 15685411
- Volume :
- 16
- Database :
- OpenAIRE
- Journal :
- Nematology
- Accession number :
- edsair.doi...........31e070d2141f77a9df67830a0263187a
- Full Text :
- https://doi.org/10.1163/15685411-00002808