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Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

Li D, Fan QH, Waite DW, Gunawardana D, George S, Kumarasinghe L - PLoS ONE (2015)

Bottom Line: The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species.The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test.Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters.

View Article: PubMed Central - PubMed

Affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, Auckland, New Zealand.

ABSTRACT
Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

No MeSH data available.


Related in: MedlinePlus

Sensitivity test of the real-time PCR assay for the identification of T. urticae.Plasmid containing ITS1 insert of T. urticae were series diluted to create calibration curves for sensitivity calculations. The standard curve built from Cq values against the log copy number (range = 107–10 copies) of ITS1 insert (n = 3). The 95% confidence intervals of the slopes were plotted with a blue line for LPMQ66 and a red line for LPMQ25. The r2 = 0.993 for LPMQ66 and 0.990 for LPMQ25 were obtained for the assay.
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pone.0131887.g001: Sensitivity test of the real-time PCR assay for the identification of T. urticae.Plasmid containing ITS1 insert of T. urticae were series diluted to create calibration curves for sensitivity calculations. The standard curve built from Cq values against the log copy number (range = 107–10 copies) of ITS1 insert (n = 3). The 95% confidence intervals of the slopes were plotted with a blue line for LPMQ66 and a red line for LPMQ25. The r2 = 0.993 for LPMQ66 and 0.990 for LPMQ25 were obtained for the assay.

Mentions: The linear dynamic range for the assay was tested on two plasmid DNAs containing the ITS1 inserts and extended from 107–10 copies of plasmid DNA. The 95% confidence limits of the linear dynamic range were plotted in Fig 1. Amplification efficiencies for both plasmids were 98% with a strong correlation coefficient (r2 = 0.993 and 0.990%) (Fig 1).


Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

Li D, Fan QH, Waite DW, Gunawardana D, George S, Kumarasinghe L - PLoS ONE (2015)

Sensitivity test of the real-time PCR assay for the identification of T. urticae.Plasmid containing ITS1 insert of T. urticae were series diluted to create calibration curves for sensitivity calculations. The standard curve built from Cq values against the log copy number (range = 107–10 copies) of ITS1 insert (n = 3). The 95% confidence intervals of the slopes were plotted with a blue line for LPMQ66 and a red line for LPMQ25. The r2 = 0.993 for LPMQ66 and 0.990 for LPMQ25 were obtained for the assay.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492583&req=5

pone.0131887.g001: Sensitivity test of the real-time PCR assay for the identification of T. urticae.Plasmid containing ITS1 insert of T. urticae were series diluted to create calibration curves for sensitivity calculations. The standard curve built from Cq values against the log copy number (range = 107–10 copies) of ITS1 insert (n = 3). The 95% confidence intervals of the slopes were plotted with a blue line for LPMQ66 and a red line for LPMQ25. The r2 = 0.993 for LPMQ66 and 0.990 for LPMQ25 were obtained for the assay.
Mentions: The linear dynamic range for the assay was tested on two plasmid DNAs containing the ITS1 inserts and extended from 107–10 copies of plasmid DNA. The 95% confidence limits of the linear dynamic range were plotted in Fig 1. Amplification efficiencies for both plasmids were 98% with a strong correlation coefficient (r2 = 0.993 and 0.990%) (Fig 1).

Bottom Line: The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species.The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test.Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters.

View Article: PubMed Central - PubMed

Affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, Auckland, New Zealand.

ABSTRACT
Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

No MeSH data available.


Related in: MedlinePlus