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Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification.

Lai GH, Chao J, Lin MK, Chang WT, Peng WH, Sun FC, Lee MS, Lee MS - Int J Mol Sci (2015)

Bottom Line: Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan.In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established.In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 40402, Taiwan. just_dfm@hotmail.com.

ABSTRACT
Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.

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Specificity of the LAMP assay for the authentication of T. formosanum (A). The LAMP reactions were performed using specifically designed LAMP primers of TF. Lane M, 1 kb of DNA marker; lane N, without plant genomic DNA; lanes 1, Taraxacum formosanum; lane 2, Taraxacum officinale; lane 3, Ixeridium laevigatum; lane 4, Youngia japonica; lane 5, Ixeris chinensis; lane 6, Emilia sonchifolia var. javanica; Sensitivity analysis of the LAMP assay for the authentication of T. formosanum (B). The LAMP reaction was performed using various quantities of genomic TF DNA as template. The upper panels represent LAMP products detected by DNA electrophoresis; the lower panels represented LAMP products detected by SYBR Green I staining and UV excitation. Lane 1, 100 ng; lane 2, 20 ng; lane 3, 1 ng; lane 4, 100 pg; lane 5, 10 pg; lane 6, 1 pg; lane 7, 100 fg; lane 8, 10 fg; lane 9, 1 fg.
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ijms-16-01562-f003: Specificity of the LAMP assay for the authentication of T. formosanum (A). The LAMP reactions were performed using specifically designed LAMP primers of TF. Lane M, 1 kb of DNA marker; lane N, without plant genomic DNA; lanes 1, Taraxacum formosanum; lane 2, Taraxacum officinale; lane 3, Ixeridium laevigatum; lane 4, Youngia japonica; lane 5, Ixeris chinensis; lane 6, Emilia sonchifolia var. javanica; Sensitivity analysis of the LAMP assay for the authentication of T. formosanum (B). The LAMP reaction was performed using various quantities of genomic TF DNA as template. The upper panels represent LAMP products detected by DNA electrophoresis; the lower panels represented LAMP products detected by SYBR Green I staining and UV excitation. Lane 1, 100 ng; lane 2, 20 ng; lane 3, 1 ng; lane 4, 100 pg; lane 5, 10 pg; lane 6, 1 pg; lane 7, 100 fg; lane 8, 10 fg; lane 9, 1 fg.

Mentions: To assess the specificity of LAMP, TF genomic DNA and genomic DNA from the five adulterant plants were used as template. The specificity of the LAMP assay for authentication of TF is shown in Figure 3A. When the LAMP primers were used to test DNA amplification, no amplification in non-targeted adulterant plant DNA was observed. However, when TF genomic DNA was present, the ladder-like LAMP DNA pattern was detected (Figure 3, lane 1). Thus the LAMP method developed in this study is able to discriminate TF from its common adulterants and this can be done rapidly under isothermal conditions within 45 min when SYBR Green dye is added. To evaluate the sensitivity of the LAMP assay when authenticating TF genomic DNA, the varying amounts of extracted TF genomic DNA were used as template DNA. The amount of TF DNA ranged from 100 ng to 1 fg. After performing the LAMP reaction, the ladder-like DNA pattern was observed even when only 1 pg of TF DNA was added to the reaction mixture (Figure 3). No LAMP product was detected by DNA electrophoresis when <1 pg of template DNA was added to the LAMP reaction. The sensitivity results were confirmed using the SYBR Green I fluorescence assay (Figure 3B, lower panel). Therefore, the sensitivity of the LAMP for authenticating TF developed in this study is 1 pg. In contrast, when traditional species-specific PCR reaction was used to detect genomic TF DNA template using two specific designed oligonucleotides F3 and B3 as PCR primer, approximately a 250 bp of PCR product was observed. As to its sensitivity, however, no presence of any PCR product was observed when <1 ng of template TF DNA was added to the PCR reaction (Figure 4); thus the sensitivity is significantly lower than that of LAMP. These results suggest that LAMP is a highly specific and sensitive method for detection of TF genomic DNA.


Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification.

Lai GH, Chao J, Lin MK, Chang WT, Peng WH, Sun FC, Lee MS, Lee MS - Int J Mol Sci (2015)

Specificity of the LAMP assay for the authentication of T. formosanum (A). The LAMP reactions were performed using specifically designed LAMP primers of TF. Lane M, 1 kb of DNA marker; lane N, without plant genomic DNA; lanes 1, Taraxacum formosanum; lane 2, Taraxacum officinale; lane 3, Ixeridium laevigatum; lane 4, Youngia japonica; lane 5, Ixeris chinensis; lane 6, Emilia sonchifolia var. javanica; Sensitivity analysis of the LAMP assay for the authentication of T. formosanum (B). The LAMP reaction was performed using various quantities of genomic TF DNA as template. The upper panels represent LAMP products detected by DNA electrophoresis; the lower panels represented LAMP products detected by SYBR Green I staining and UV excitation. Lane 1, 100 ng; lane 2, 20 ng; lane 3, 1 ng; lane 4, 100 pg; lane 5, 10 pg; lane 6, 1 pg; lane 7, 100 fg; lane 8, 10 fg; lane 9, 1 fg.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4307320&req=5

ijms-16-01562-f003: Specificity of the LAMP assay for the authentication of T. formosanum (A). The LAMP reactions were performed using specifically designed LAMP primers of TF. Lane M, 1 kb of DNA marker; lane N, without plant genomic DNA; lanes 1, Taraxacum formosanum; lane 2, Taraxacum officinale; lane 3, Ixeridium laevigatum; lane 4, Youngia japonica; lane 5, Ixeris chinensis; lane 6, Emilia sonchifolia var. javanica; Sensitivity analysis of the LAMP assay for the authentication of T. formosanum (B). The LAMP reaction was performed using various quantities of genomic TF DNA as template. The upper panels represent LAMP products detected by DNA electrophoresis; the lower panels represented LAMP products detected by SYBR Green I staining and UV excitation. Lane 1, 100 ng; lane 2, 20 ng; lane 3, 1 ng; lane 4, 100 pg; lane 5, 10 pg; lane 6, 1 pg; lane 7, 100 fg; lane 8, 10 fg; lane 9, 1 fg.
Mentions: To assess the specificity of LAMP, TF genomic DNA and genomic DNA from the five adulterant plants were used as template. The specificity of the LAMP assay for authentication of TF is shown in Figure 3A. When the LAMP primers were used to test DNA amplification, no amplification in non-targeted adulterant plant DNA was observed. However, when TF genomic DNA was present, the ladder-like LAMP DNA pattern was detected (Figure 3, lane 1). Thus the LAMP method developed in this study is able to discriminate TF from its common adulterants and this can be done rapidly under isothermal conditions within 45 min when SYBR Green dye is added. To evaluate the sensitivity of the LAMP assay when authenticating TF genomic DNA, the varying amounts of extracted TF genomic DNA were used as template DNA. The amount of TF DNA ranged from 100 ng to 1 fg. After performing the LAMP reaction, the ladder-like DNA pattern was observed even when only 1 pg of TF DNA was added to the reaction mixture (Figure 3). No LAMP product was detected by DNA electrophoresis when <1 pg of template DNA was added to the LAMP reaction. The sensitivity results were confirmed using the SYBR Green I fluorescence assay (Figure 3B, lower panel). Therefore, the sensitivity of the LAMP for authenticating TF developed in this study is 1 pg. In contrast, when traditional species-specific PCR reaction was used to detect genomic TF DNA template using two specific designed oligonucleotides F3 and B3 as PCR primer, approximately a 250 bp of PCR product was observed. As to its sensitivity, however, no presence of any PCR product was observed when <1 ng of template TF DNA was added to the PCR reaction (Figure 4); thus the sensitivity is significantly lower than that of LAMP. These results suggest that LAMP is a highly specific and sensitive method for detection of TF genomic DNA.

Bottom Line: Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan.In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established.In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 40402, Taiwan. just_dfm@hotmail.com.

ABSTRACT
Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.

Show MeSH
Related in: MedlinePlus