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. 2023 Sep 29;28(19):6872.
doi: 10.3390/molecules28196872.

Rapid Discrimination of Panax quinquefolium and Panax ginseng Using the Proofman-Duplex-LMTIA Technique

Xiaodong Zhang  1 , Zongding Li  1 , Yaoxuan Zhang  2 , Dandan Xu  3 , Liang Zhang  1 , Fugang Xiao  1 , Deguo Wang  1
Affiliations

Rapid Discrimination of Panax quinquefolium and Panax ginseng Using the Proofman-Duplex-LMTIA Technique

Xiaodong Zhang et al. Molecules. .

Abstract

This study aims to establish a rapid identification method based on the Proofman-LMTIA technique for distinguishing between Panax quinquefolium and Panax ginseng. By targeting specific 18S rDNA sequences, suitable primers and Proofman probes labeled FAM or JOE were designed for LMTIA. Initially, single-species-primer Proofman-LMTIA assays were performed separately for each ginseng type to optimize reaction temperature, assess sensitivity and specificity, and determine the detection limit. Subsequently, both sets of primers and their corresponding probes were combined in the same reaction system to further optimize reaction conditions, evaluate sensitivity, and assess stability. Finally, the developed Proofman-duplex-LMTIA technique was employed to detect P. quinquefolium and P. ginseng slices available in the market. Single-plex Proofman-LMTIA assays revealed that the optimal reaction temperature for both P. quinquefolium and P. ginseng was 62 °C. The sensitivity was as low as 1 pg/μL, with a detection limit of 0.1%, and both showed excellent specificity. The optimal temperature for Proofman-duplex-LMTIA assays was 58 °C. This method could simultaneously identify P. quinquefolium and P. ginseng. Testing 6 samples of P. ginseng and 11 samples of P. quinquefolium from the market resulted in a 100% positive rate for all samples. This study successfully established a rapid, simple, sensitive, and specific Proofman-duplex-LMTIA identification method for P. quinquefolium and P. ginseng. It provides an effective means for quality control of P. quinquefolium, P. ginseng, and related products.

Keywords: Panax ginseng; Panax quinquefolium; Proofman probe; ladder melting temperature isothermal amplification; rapid identification.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Alignment results of 18S rDNA differential sequences among P. quinquefolium, P. ginseng and their relatives.
Figure 2
Figure 2
Proofman−LMTIA reaction for P. quinquefolium and P. ginseng at different temperatures. (a–d): Proofman−LMTIA reaction for P. quinquefolium at 61 °C, 62 °C, 63 °C, and 64 °C, respectively. Purple curve: genomic DNA of P. quinquefolium; green curve: DEPC−treated water. (e–h): Proofman-LMTIA reaction for P. ginseng at 61 °C, 62 °C, 63 °C, and 64 °C, respectively. Red curve: genomic DNA of P. ginseng; blue curve: DEPC−treated water.
Figure 3
Figure 3
Proofman−LMTIA reaction for P. quinquefolium and P. ginseng at different concentration of template. (a) 1–5: genomic DNA concentration of P. quinquefolium was 10 ng/μL, 1 ng/μL, 100 pg/μL, 10 pg/μL and 1 pg/μL. 6: DEPC−treated water. (b) 1–5: genomic DNA concentration of P. ginseng was 10 ng/μL, 1 ng/μL, 100 pg/μL, 10 pg/μL, and 1 pg/μL. 6: DEPC−treated water.
Figure 4
Figure 4
Proofman−LMTIA specific reaction for P. quinquefolium and P. ginseng. (a) 1: 10 ng/μL of genomic DNA of P. quinquefolium; 2: 10 ng/μL of genomic DNA of P. ginseng; (b) 1: 10 ng/μL of genomic DNA of P. ginseng; 2: 10 ng/μL of genomic DNA of P. quinquefolium.
Figure 5
Figure 5
Proofman−LMTIA reaction for simulated adulteration experiment of P. quinquefolium and P. ginseng. (a) 1–6: 100%, 20%, 10%, 5%, 1%, and 0.1% of genomic DNA of P. quinquefolium in the mixture of P. quinquefolium and P. ginseng; 7: DEPC−treated water; (b) 1–6: 100%, 20%, 10%, 5%, 1%, and 0.1% of genomic DNA of P. ginseng in the mixture of P. quinquefolium and P. ginseng; 7: DEPC−treated water.
Figure 6
Figure 6
Proofman−duplex−LMTIA temperature optimization reaction for simultaneous detection of P. quinquefolium and P. ginseng. (a) 58 °C; (b) 60 °C; (c) 62 °C; and (d) 64 °C. 1: genomic DNA of P. ginseng; 2: genomic DNA of P. quinquefolium; 3: DEPC−treated water.
Figure 7
Figure 7
Proofman−duplex−LMTIA sensitivity detection for simultaneous detection of P. quinquefolium and P ginseng. (a) Proofman-duplex-LMTIA sensitivity detection for P. quinquefolium. 1–5: genomic DNA concentration of P. quinquefolium was 10 ng/μL, 1 ng/μL, 100 pg/μL, 10 pg/μL, and 1 pg/μL, respectively. 6: DEPC−treated water. (b) Proofman-duplex-LMTIA sensitivity detection for P. ginseng. 1–5: genomic DNA concentration of P. quinquefolium was 10 ng/μL, 1 ng/μL, 100 pg/μL, 10 pg/μL, and 1 pg/μL, respectively. 6: DEPC−treated water.
Figure 8
Figure 8
Proofman−duplex−LMTIA stability detection for simultaneous detection of P. quinquefolium and P. ginseng. 1: genomic DNA of P. ginseng; 2: genomic DNA of P. quinquefolium; 3: DEPC−treated water.
Figure 9
Figure 9
Simultaneously testing P. quinquefolium and P. ginseng slices in the market using Proofman−duplex−LMTIA. 1: genomic DNA of six P. ginseng slices; 2: genomic DNA of eleven P. quinquefolium slices; and 3: DEPC−treated water.
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