LMTIA primer group and Proofman probe for double detection of Schisandra chinensis, kit, application and detection method

Abstract

The invention relates to an LMTIA primer group and a Proofman probe for double detection of Schisandra chinensis, a kit, application and a detection method, and belongs to the technical field of biomolecular detection. The LMTIA primer group and the Proofman probe for double detection of Schisandra chinensis are designed by taking ITS1 of Schisandra chinensis and Schisandra chinensis as target sequences. The invention also discloses a preparation method of the LMTIA primer group and the Proofman probe for double detection of Schisandra chinensis and Schisandra chinensis. The LMTIA primer group comprises WuWZ-F1 as shown in a sequence SEQ ID NO 1, WuWZ-B13 as shown in a sequence SEQ ID NO 2, WuWZ-F3 as shown in a sequence SEQ ID NO 3, WuWZ-LF as shown in a sequence SEQ ID NO 4 and NWuWZ-LF as shown in a sequence SEQ ID NO 5; the Proofman probe comprises WuWZProbe as shown in a sequence SEQ ID NO (Sequence Identity Number 6) and NWuWZProbe as shown in a sequence SEQ ID NO (Sequence Identity Number 7). The LMTIA primer group and the Proofman probe for double detection of Schisandra chinensis have good specificity and sensitivity, the detection speed is high, and accurate detection of Schisandra chinensis components in a sample can be realized under a constant temperature condition.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomolecule detection, and particularly relates to a LMTIA primer set and Proofman probe for double detection of Schisandra sphenanthera and Schisandra chinensis, a kit, application and detection method. BACKGROUND

[0002] The 2025 edition of Chinese Pharmacopoeia stipulates that Schisandra chinensis (Turcz.) Baill. is the dried mature fruit of Schisandra chinensis (Turcz.) Baill. of Magnoliaceae, which is commonly known as "North Schisandra". Its functions are astringent, tonifying Qi, and it can also be used for treating chronic cough, asthma, dream and seminal emission, frequent urination, chronic diarrhea, self-hemorrhoids, thirst, internal heat, and heart palpitations. The Pharmacopoeia also stipulates that Schisandra sphenanthera Rehd. et Wils. is the dried mature fruit of Schisandra sphenanthera Rehd. et Wils. of Magnoliaceae, and its functions, indications and uses are the same as those of North Schisandra. Modern research shows that the main chemical components of South and North Schisandra are lignans, polysaccharides, volatile oils, organic acids, terpenes, amino acids, vitamins and mineral elements, but there are significant differences in the types and contents of specific chemical components between the two, such as schisandrin A, schisandrin B, schisantherin A, deoxyschizandrin, schisantherin B, schisantherin C, and total lignans, which have statistically significant differences (p<0.01) in North and South Schisandra and their pericarps, and the total lignan content in North Schisandra is significantly higher than that in South Schisandra. Clinical research shows that the two have different effects: North Schisandra is better at nourishing five internal organs and soothing the heart and mind, while South Schisandra is best at dispersing external pathogens and relieving cold and asthma.

[0003] In addition, due to the differences in chemical components between South and North Schisandra, the price of North Schisandra is about 20% higher than that of South Schisandra in the market, and this price difference prompts some merchants to use low-priced South Schisandra to substitute or adulterate high-priced North Schisandra in the production and processing of medicinal materials, medicines, food and beverages, which seriously damages the health rights and interests and economic interests of consumers.

[0004] To address this issue, the 2025 edition of the Chinese Pharmacopoeia provides methods for identifying Schisandra sphenanthera and S. chinensis, including property identification, microscopic identification, thin-layer chromatography identification, and high-performance liquid chromatography (HPLC) determination. However, these methods have some limitations in practical application, such as the subjectivity of property identification, the need for equipment in microscopic identification, the dependence on standard samples in thin-layer chromatography, and the requirement for expensive instruments and professional operation skills in HPLC method. Given the morphological similarity between S. sphenanthera and S. chinensis and the inability to distinguish them by the naked eye after being processed into powder or traditional Chinese medicine, it is particularly urgent to develop a rapid, low-cost, and equipment-free method for identifying S. sphenanthera and S. chinensis suitable for the general public.

[0005] The invention patent with the application publication number CN107619878A discloses a Schisandra molecular identity card and an identification method. The identification method includes using the genomic DNA of the sample to be tested as a template to perform PCR amplification on the molecular identity card to obtain an amplification product; sequencing the amplification product, splicing, and removing the primer region to obtain an amplification fragment; and detecting whether the molecular identity card exists in the amplification fragment. This identification method can accurately distinguish S. sphenanthera and S. chinensis, but requires a long detection time. SUMMARY

[0006] The first object of the present application is to provide a method for detecting S. sphenanthera and S. chinensis based on double Proofman-LMTIA to solve the technical problem of the need for complex equipment and a long detection time in the prior art.

[0007] The second object of the present application is to provide a LMTIA primer set and Proofman probe for double detection of S. sphenanthera and S. chinensis.

[0008] The third object of the present application is to provide a kit for authenticity detection of S. sphenanthera and S. chinensis.

[0009] The fourth object of the present application is to provide an application of a LMTIA primer set and Proofman probe for double detection of S. sphenanthera and S. chinensis.

[0010] To achieve the above objects, the technical solution adopted by the present application is as follows:

[0011] The LMTIA primer set and Proofman probe for double detection of S. sphenanthera and S. chinensis are designed based on the ITS1 of S. sphenanthera and S. chinensis as the target sequence;

[0012] The LMTIA primer set includes: WuWZ-F1 as shown in sequence SEQ ID NO 1, WuWZ-B13 as shown in sequence SEQ ID NO 2, WuWZ-F3 as shown in sequence SEQ ID NO 3, WuWZ-LF as shown in sequence SEQ ID NO 4, and NWuWZ-LF as shown in sequence SEQ ID NO 5;

[0013] The Proofman probes include WuWZProbe as shown in sequence SEQ ID NO 6 and NWuWZProbe as shown in sequence SEQ ID NO 7.

[0014] Furthermore, the 5' end of the WuWZProbe is labeled with a BHQ2 quenching group, and the 3' end is labeled with a 6-FAM fluorescent group; the 5' end of the NWuWZProbe is labeled with a BHQ2 quenching group, and the 3' end is labeled with a 6-JOE fluorescent group.

[0015] A kit for the authenticity testing of Schisandra chinensis (Southern and Northern varieties) includes the aforementioned LMTIA primer set and Proofman probe.

[0016] Applications of LMTIA primer sets and Proofman probes for dual detection of Schisandra chinensis and Schisandra spp., or kits for authenticity testing of Schisandra chinensis and Schisandra spp. in the detection of seeds, seedlings, vines, processed slices, and food and pharmaceuticals containing Schisandra chinensis and Schisandra spp.

[0017] The detection method for Schisandra chinensis based on dual Proofman-LMTIA includes the following steps: taking DNA from the sample to be tested, preparing the Proofman-LMTIA reaction system, and observing the fluorescence signal or amplification curve after isothermal amplification in a real-time quantitative PCR system or a constant-temperature metal bath to detect the presence of Schisandra chinensis and / or Schisandra chinensis; the Proofman-LMTIA reaction system includes the above-mentioned LMTIA primer set and Proofman probe.

[0018] Furthermore, the molar ratio of WuWZ-F1:WuWZ-B13:WuWZ-F3:WuWZ-LF:NWuWZ-LF:WuWZ-Probe:NWuWZ-Probe in the Proofman-LMTIA reaction system is 8:8:8:8:2:2:15:15. In the sample detection tube after the reaction is complete, if a fluorescence signal is detected in the green fluorescence channel (FAM) or an exponential amplification curve appears in the amplification result image, the sample contains Schisandra chinensis (Northern Schisandra); if a fluorescence signal is detected in the yellow fluorescence channel (JOE) or an exponential amplification curve appears in the amplification result image, the sample contains Schisandra chinensis (Southern Schisandra); if no fluorescence signal is detected in the green fluorescence channel (FAM) or no exponential amplification curve appears in the amplification result image, the sample does not contain Schisandra chinensis (Northern Schisandra); if no fluorescence signal is detected in the yellow fluorescence channel (JOE) or no exponential amplification curve appears in the amplification result image, the sample does not contain Schisandra chinensis (Southern Schisandra).

[0019] Furthermore, the isothermal amplification procedure is as follows: 60℃, 40 cycles, fluorescence signal acquisition every 30 seconds, for a total of 40 fluorescence signal acquisitions.

[0020] Furthermore, the Proofman-LMTIA reaction system has a volume of 10 μL and contains 5 μL of 2×Mix premix, 0.4 μL of ultra-fidelity DNA polymerase, 0.16 μL of WuWZ-F1, 0.16 μL of WuWZ-B13, 0.16 μL of WuWZ-F3, 0.04 μL of WuWZ-LF, 0.04 μL of NWuWZ-LF, 0.3 μL of WuWZProbe, 0.3 μL of NWuWZProbe, 2 μL of genomic DNA template, and 1.44 μL of water.

[0021] The beneficial effects of this invention are:

[0022] The LMTIA primer set and Proofman probe of this invention for dual detection of Schisandra chinensis and Schisandra spp. exhibit excellent specificity and sensitivity, rapid detection speed, and accurate detection of Schisandra chinensis components in samples under isothermal conditions. The absolute sensitivities of the LMTIA primer set and Proofman probe for Schisandra chinensis and Schisandra spp. of this invention reach 10 fg / μL and 1 fg / μL, respectively, and the detection limits for Schisandra chinensis and Schisandra spp. of this invention reach 1% and 0.1%, respectively. The LMTIA primer set and Proofman probe of this invention can be used not only for detecting raw materials such as seeds, seedlings, vines, and processed slices of Schisandra chinensis and Schisandra spp., but also for the authenticity verification of end products such as food, pharmaceuticals, and beverages containing Schisandra chinensis components.

[0023] The present invention provides a simple and rapid detection method for Schisandra chinensis based on dual Proofman-LMTIA. Under isothermal conditions, amplification of about 20 minutes can achieve rapid and accurate detection of Schisandra chinensis components in the sample, and it has good repeatability and high stability.

[0024] This invention, based on the sequence characteristics of ITS1, utilizes DNA barcoding molecular identification to develop a rapid detection method for Schisandra chinensis (Southern and Northern varieties) and their processed products. The ITS1 sequence, due to its multiple copy nature in the plant genome, provides a reliable molecular marker for addressing the partial degradation of genomic DNA during medicinal material processing. This invention selects the ITS1 sequence as the detection target and, through carefully designed primers and probes combined with Proofman-LMTIA technology, achieves efficient and authentic identification of Schisandra chinensis (Southern and Northern varieties) and their processed products. Attached Figure Description

[0025] Figure 1 This is a diagram showing the ITS1 sequence alignment results of Schisandra chinensis (Northern and Southern varieties) in Example 1.

[0026] Figure 2 This is a PCR result diagram of the genomic DNA of Schisandra chinensis and Schisandra sibirica in Example 1, where lane M: DNA molecular weight standard; lanes 1-2: Schisandra chinensis; lanes 3-4: Schisandra sibirica;

[0027] Figure 3 This image shows the enzyme digestion results of the plasmid DNA from *Schisandra chinensis* and *Schisandra sibiricum* in Example 1. Lane M: DNA molecular weight standard; Lanes 1, 3, and 5: EcoRI digestion results of the *Schisandra sibiricum* pMD19-ScITS1 plasmid; Lanes 2, 4, and 6: *Schisandra sibiricum* pMD19-ScITS1 plasmid control; Lanes 7-10: EcoRI digestion results of the *Schisandra chinensis* pMD19-SsITS1 plasmid.

[0028] Figure 4 This is a diagram showing the amplification results of the F1 primer set of Schisandra chinensis plasmid DNA in Example 1 using the dye method.

[0029] Figure 5 This is a diagram showing the amplification results of the F2 primer set of Schisandra chinensis plasmid DNA in Example 1 using the dye method.

[0030] Figure 6 This is a diagram showing the amplification results of the F3 primer set of Schisandra chinensis plasmid DNA in Example 1 using the dye method.

[0031] Figure 7 This is a diagram showing the amplification results of the F1 primer set of Schisandra chinensis plasmid DNA using dye method in Example 1;

[0032] Figure 8This is a diagram showing the amplification results of the F2 primer set of Schisandra chinensis plasmid DNA in Example 1 using the dye method.

[0033] Figure 9 This is a diagram showing the amplification results of the F3 primer set of Schisandra chinensis plasmid DNA using dye method in Example 1;

[0034] Figure 10 This is a graph showing the temperature optimization results of the Schisandra chinensis plasmid DNA Proofman-LMTIA in Example 2;

[0035] Figure 11 This is a diagram showing the specificity detection results of the Schisandra chinensis plasmid DNA Proofman-LMTIA in Example 3;

[0036] Figure 12 This is a graph showing the Proofman-LMTIA sensitivity detection results of Schisandra chinensis plasmid DNA in Example 4;

[0037] Figure 13 The figure shows the Proofman-LMTIA detection limit test results of Schisandra chinensis plasmid DNA in Example 5;

[0038] Figure 14 This is a diagram showing the Proofman-LMTIA temperature optimization results of the Schisandra chinensis plasmid DNA in Example 6;

[0039] Figure 15 This is a diagram showing the Proofman-LMTIA specificity detection results of the Schisandra chinensis plasmid DNA in Example 7;

[0040] Figure 16 This is a graph showing the Proofman-LMTIA sensitivity detection results of the Schisandra chinensis plasmid DNA in Example 8;

[0041] Figure 17 The image shows the detection results of the Proofman-LMTIA detection limit of the Schisandra chinensis plasmid DNA in Example 9;

[0042] Figure 18 The figure shows the temperature optimization results of the dual Proofman-LMTIA method for Schisandra chinensis plasmid DNA in Example 10.

[0043] Figure 19 This is a diagram showing the results of dual Proofman-LMTIA specific detection of Schisandra chinensis and Schisandra sibiricum plasmid DNA in Example 11;

[0044] Figure 20 This is a graph showing the dual Proofman-LMTIA sensitivity detection results of Schisandra chinensis and Schisandra sibiricum plasmid DNA in Example 12;

[0045] Figure 21 The image shows the detection results of the dual Proofman-LMTIA detection limit of Schisandra chinensis plasmid DNA in Example 13;

[0046] Figure 22 This is a diagram showing the temperature optimization results of the dual Proofman-LMTIA method for Schisandra chinensis plasmid DNA in Example 14.

[0047] Figure 23 This is a graph showing the dual Proofman-LMTIA sensitivity detection results of Schisandra chinensis plasmid DNA in Example 15;

[0048] Figure 24 The image shows the detection results of the dual Proofman-LMTIA detection limit of the Schisandra chinensis plasmid DNA in Example 16;

[0049] Figure 25 The image shows the detection results of the dual Proofman-LMTIA limit of detection for Schisandra chinensis and Schisandra chinensis slices in Example 17;

[0050] Figure 26 The image shows the dual Proofman-LMTIA test results of the medicine containing Schisandra chinensis in Example 18. Detailed Implementation

[0051] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0052] All reagents used in this invention are commonly used molecular biology experimental reagents, and all instruments are standard equipment used in molecular biology laboratories. All primers were synthesized by Shanghai Jierui Biotechnology Co., Ltd., and probes were synthesized in our laboratory. Unless otherwise specified, the methods used in the embodiments of this invention are conventional molecular biology methods.

[0053] The GPV8 ultra-fidelity DNA polymerase concentration was 2 U / μL, purchased from Anhui Global Gene Technology Co., Ltd. The universal plant genomic DNA extraction kit (EE112-02) was purchased from Beijing TransGen Biotech Co., Ltd. Max DNA Polymerase (2×) premix was purchased from Baori Biotechnology (Beijing) Co., Ltd.; Mix premix and 2×Mix premix were purchased from Dege Biotechnology (Shandong) Co., Ltd.

[0054] Example 1

[0055] 1. Primer and probe design

[0056] The ITS1 sequences of Schisandra chinensis (southern and northern varieties) were compared and analyzed using DNMAN 10 software, accurately identifying the regions of difference between the two. The results are as follows: Figure 1 As shown in the figure. Subsequently, Oligo 7 software was used to identify regions where the melting temperature Tm exhibits a trapezoidal or semi-trapezoidal shape based on the characteristic differential sequences in the ITS1 sequences of Schisandra chinensis and Schisandra dasycarpus. Following the principles of LMTIA primer design, LMTIA primers and Proofman probes were designed using the online primer design software Primer 3PLUS (http: / / www.primer3plus.com). After multiple rounds of screening and optimization, the following LMTIA primer sets and Proofman probe sequences were obtained, as shown in Table 1.

[0057] Table 1 LMTIA primer set and Proofman probe

[0058]

[0059]

[0060] 2. Extraction and detection of genomic DNA from Schisandra chinensis (Southern and Northern varieties)

[0061] Dried Schisandra chinensis (both southern and northern varieties) were ground into powder using a traditional Chinese medicine pulverizer. 100 mg of the sample was weighed, and genomic DNA was extracted using a universal plant genomic DNA extraction kit. After extraction, the concentration and purity of the genomic DNA were determined using a Nanodrop One (Thermo Fisher Scientific, USA) nucleic acid and protein analyzer. 260 / A 280 DNA with a pH of 1.6-2.0 can be used for the next step of detection. The extracted genomic DNA should be stored at -20°C for later use.

[0062] 3. Preparation of pMD19T-ScITS1 plasmid from Schisandra chinensis and pMD19T-SsITS1 plasmid from Schisandra chinensis

[0063] In a clean bench, add the following reagent to a 200 μL PCR tube: 25 μL of... The following solutions were used: Max DNA Polymerase (2×) premix, 2 μL of 10 μmol / L ITS-2F, 2 μL of 10 μmol / L ITS-3R, 4 μL of genomic DNA, and 17 μL of ddH2O. The reaction conditions were (98℃ for 10 s, 53℃ for 5 s, 72℃ for 10 s) × 30 cycles. After the PCR reaction, an A-tailing reaction was performed.

[0064] The PCR products were separated using agarose gel electrophoresis, and the results are as follows: Figure 2 As shown, after electrophoresis, the gel was cut, recovered, and ligated into the pMD19T vector. *E. coli* DH5α was transformed using the heat shock method, then plated on AIX plates supplemented with ampicillin, IPTG, and X-gal, and incubated at 37°C. After picking and shaking, plasmids were extracted. EcoRI digestion confirmed successful insertion of the target fragment into the vector, as shown in the results. Figure 3 As shown in the image. Finally, the bacterial culture with correctly identified clones was sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing. After sequencing, the pMD19T-ScITS1 plasmid from *Schisandra chinensis* and the pMD19T-SsITS1 plasmid from *Schisandra sibirica* were obtained. The plasmids were subsequently used as primers for screening and as positive controls for DNA templates constructed using the Proofman-LMTIA method and the kit.

[0065] 4. Screening of LMTIA primers for Schisandra chinensis (Northern and Southern varieties)

[0066] 4.1 Schisandra chinensis LMTIA primer screening: In a clean bench, the following reagents were added to a 100 μL PCR tube: 5 μL of Mix premix, 0.16 μL of 10 μmol / L WuWZ-F, 0.16 μL of 10 μmol / L WuWZ-B, 0.04 μL of 10 μmol / L NWuWZ-LF, 2 μL of 10 ng / μL Schisandra chinensis plasmid pMD19T-ScITS1, and 2.64 μL of ddH2O. The detection was performed using a Gentier 96E fully automated medical PCR analysis system. The reaction temperature was set to 52℃, 54℃, 56℃, and 58℃, the cycle number was set to 40, and fluorescence signals were collected every 90 seconds for a total of 40 collections. The experimental group contained Schisandra chinensis plasmid DNA at a concentration of 10 ng / μL, and the negative control was DEPC-treated water.

[0067] 4.2. Schisandra chinensis LMTIA primer screening: The difference from Schisandra chinensis LMTIA primer screening is that 0.04 μL of 10 μmol / L NWuWZ-LF is replaced with 0.04 μL of 10 μmol / L WuWZ-LF; and 2 μL of 10 ng / μL Schisandra chinensis plasmid pMD19T-ScITS1 is replaced with 2 μL of 10 ng / μL Schisandra chinensis plasmid pMD19T-SsITS1. The experimental group contains 10 ng / μL Schisandra chinensis plasmid DNA.

[0068] If a fluorescence signal is detected in the green fluorescence (SYBR I) channel and an exponential amplification curve appears in the amplification result graph, it indicates that the extracted genomic DNA or plasmid DNA has been successfully amplified. If no fluorescence signal is detected in the green fluorescence (SYBR I) channel and no exponential amplification curve appears in the amplification result graph, it indicates that the extracted genomic DNA or plasmid DNA has failed to amplify. The criteria for primer selection are the fewest initial amplification cycles and the highest amplification efficiency. In this embodiment, a real-time quantitative PCR system is used for isothermal amplification reactions. In other embodiments, a constant-temperature metal bath or other isothermal equipment can also be used.

[0069] The primer detection results of Schisandra chinensis are as follows: Figure 4 , Figure 5 , Figure 6 As shown in the figure, the curves marked with temperature correspond to the *Schisandra chinensis* plasmid DNA, and the curves marked with H2O are negative controls. Observation of the amplification curves shows that the negative controls showed no amplification. For primer set F1, at 58℃, 56℃, 54℃, and 52℃, the *Schisandra chinensis* plasmid DNA began to amplify from the 12.85th, 16.35th, 22.70th, and 29.55th cycles, respectively; for primer set F2, at 58℃, 56℃, and 54℃, the *Schisandra chinensis* plasmid DNA began to amplify from the 15.04th, 18.02nd, and 25.58th cycles, respectively; and for primer set F3, at 58℃, 56℃, 54℃, and 52℃, the *Schisandra chinensis* plasmid DNA began to amplify from the 10.50th, 12.63rd, 17.35th, and 25.51st cycles, respectively. Therefore, the F3 primer set had the lowest cycle number and the highest amplification efficiency at 58℃, and was selected for subsequent detection of Schisandra chinensis.

[0070] The primer detection results of Schisandra chinensis are as follows: Figure 7 , Figure 8 , Figure 9 As shown in the figure, the curves marked with temperature correspond to the *Schisandra chinensis* plasmid DNA, and the curves marked with H2O are negative controls. Observation of the amplification curves shows that the negative controls showed no amplification. For primer set F1, at 58℃, 56℃, 54℃, and 52℃, the *Schisandra chinensis* plasmid DNA began to amplify from the 9.02, 10.76, 14.02, and 21.03th cycles, respectively; for primer set F2, at 58℃, 56℃, and 54℃, the plasmid DNA began to amplify from the 9.50, 10.50, 14.31, and 24.29th cycles, respectively; and for primer set F3, at 58℃, 56℃, 54℃, and 52℃, the plasmid DNA began to amplify from the 11.64, 11.52, 17.89, and 26.72nd cycles, respectively. Primer set F1 showed the lowest initial cycle number and highest amplification efficiency at 58℃. Therefore, the F1 primer set was selected for subsequent detection of Schisandra chinensis.

[0071] Example 2

[0072] Single-weight Proofman-LMTIA temperature optimization test of Schisandra chinensis

[0073] In a clean bench, add the following reaction mixture to a 100 μL PCR tube: 5 μL 2×Mix premix, 0.4 μL 2 U / μL GPV8 high-fidelity DNA polymerase, 0.16 μL 10 μmol / L WuWZ-F3, 0.16 μL 10 μmol / L WuWZ-B13, 0.04 μL 10 μmol / L WuWZ-LF, 0.3 μL 10 μmol / L WuWZProbe, 2 μL Schisandra chinensis pMD19T-ScITS1 plasmid DNA template, and 1.94 μL DEPC-treated water. The Gentier 96E fully automated medical PCR analysis system was used for detection. Reaction temperatures were 59℃, 61℃, 63℃, and 65℃, with a cycle count of 40. Fluorescence signals were collected every 30 seconds for a total of 40 collections. The test sample was pMD19T-ScITS1 plasmid DNA from *Schisandra chinensis*, and the negative control was DEPC-treated water. Each experiment was performed in duplicate. The results were statistically analyzed and observed using the Gentier 96E fully automated medical PCR analysis system software. The temperature optimization test results are shown below. Figure 10 As shown in the figure, the curves marked with temperature correspond to the pMD19T-ScITS1 plasmid DNA from *Schisandra chinensis*. At reaction temperatures of 65℃, 63℃, 61℃, and 59℃, the pMD19T-ScITS1 plasmid DNA from *Schisandra chinensis* began amplification from the 23.09th, 20.51st, 17.62nd, and 24.25th cycles, respectively. The negative control H2O showed no amplification. The reaction temperature of 61℃ resulted in the lowest number of amplification cycles; therefore, 61℃ was selected as the optimal reaction temperature for the single Proofman-LMTIA reaction of *Schisandra chinensis*.

[0074] Example 3

[0075] Proofman-LMTIA specificity test for Schisandra chinensis

[0076] Using 10 ng / μL of *Schisandra chinensis* pMD19T-ScITS1 plasmid DNA as a positive control and 10 ng / μL of *Schisandra suspensa* pMD19T-SsITS1 plasmid DNA and H2O as negative controls, the optimized Proofman-LMTIA reaction system established in Example 2 was used. The reaction was performed using a Gentier 96E fully automated medical PCR analysis system at 61°C for 40 cycles. Fluorescence signals were collected every 30 seconds for a total of 40 collections. Each reaction was performed in quadruplicate. The results were statistically analyzed and visualized using the Gentier 96E fully automated medical PCR analysis system. Specificity test results are as follows: Figure 11 As shown in the figure, the exponential amplification curve corresponds to the Schisandra chinensis plasmid DNA, and the baseline curve corresponds to the Schisandra chinensis plasmid DNA and H2O. The Schisandra chinensis pMD19T-ScITS1 plasmid has good amplification efficiency, while the Schisandra chinensis pMD19T-SsITS1 plasmid and the negative control have no amplification, indicating that the Proofman-LMTIA reaction system established in Example 2 has good specificity for the Schisandra chinensis pMD19T-ScITS1 plasmid DNA.

[0077] Example 4

[0078] Single Proofman-LMTIA Sensitivity Test of Schisandra chinensis

[0079] The extracted pMD19T-ScITS1 plasmid DNA from Schisandra chinensis was diluted to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL, respectively, with H2O as a negative control. This was added to the optimized Proofman-LMTIA reaction system described in Example 2. Two parallel samples were set for each reaction. The temperature was set at 61℃, and fluorescence signals were collected every 30 seconds for a total of 40 collections. Sensitivity test results are as follows: Figure 12 As shown, 10 ng / μL, 1 ng / μL, and 100 pg / μL of *Schisandra chinensis* pMD19T-ScITS1 plasmid DNA all showed good amplification, while 10 pg / μL and 1 pg / μL of *Schisandra chinensis* pMD19T-ScITS1 plasmid DNA and H2O showed no amplification. Therefore, the sensitivity of this method is 100 pg / μL.

[0080] Example 5

[0081] Single Proofman-LMTIA detection limit test of Schisandra chinensis

[0082] The extracted pMD19T-ScITS1 plasmid DNA from *Schisandra chinensis* was mixed with pMD19T-SsITS1 plasmid DNA from *Schisandra spp.* at concentrations of 0%, 0.1%, 1.0%, 5.0%, 10%, 20%, and 100%, respectively. These mixed samples were then added to the optimized Proofman-LMTIA reaction system described in Example 2. Detection was performed using a Gentier 96E fully automated medical PCR analysis system, with H2O as a negative control. Each experiment was repeated twice. The temperature was set at 61°C, and fluorescence signals were collected every 30 seconds for a total of 40 collections. The detection limit test results are as follows: Figure 13 As shown in the figure, the curves marked with 100%, 20%, 10%, 5%, 1%, and 0.1% correspond to mixed samples with pMD19T-ScITS1 plasmid DNA volumes of 100%, 20%, 10%, 5.0%, 1.0%, and 0.1%, respectively. The baseline corresponds to the sample with 0% pMD19T-SsITS1 plasmid DNA volume and the negative control DEPC-treated water. Observation of the amplification curves shows that pMD19T-ScITS1 plasmid DNA from *Schisandra chinensis* was successfully amplified in the mixed samples with 100%, 20%, 10%, 5%, 1%, and 0.1% concentrations, while no amplification occurred in the 0% mixed sample and the negative control. Therefore, the detection limit of this method is 0.1%.

[0083] Example 6

[0084] Single-weight Proofman-LMTIA temperature optimization test of Schisandra chinensis

[0085] In a clean bench, add the following reaction mixture to a 100 μL PCR tube: 5 μL 2×Mix premix, 0.4 μL 2 U / μL GPV8 high-fidelity DNA polymerase, 0.16 μL 10 μmol / L primer WuWZ-F1, 0.16 μL 10 μmol / L WuWZ-B13, 0.04 μL 10 μmol / L primer NWuWZ-LF, 0.3 μL 10 μmol / L NWuWZProbe, 2 μL Schisandra chinensis pMD19T-SsITS1 plasmid DNA template, and 1.94 μL DEPC-treated water. The Gentier 96E fully automated medical PCR analysis system was used for detection. Reaction temperatures were 59℃, 61℃, 63℃, and 65℃, with a cycle count of 40. Fluorescence signals were collected every 30 seconds for a total of 40 collections. The test sample was *Schisandra chinensis* pMD19T-SsITS1 plasmid DNA, and the negative control was H2O. Each experiment was performed in duplicate. The results were statistically analyzed and observed using the Gentier 96E fully automated medical PCR analysis system software. The temperature optimization test results are shown below. Figure 14 As shown in the figure, the curves marked with temperature correspond to the pMD19T-SsITS1 plasmid DNA from *Schisandra chinensis*, with H2O serving as the negative control. No amplification occurred at a reaction temperature of 65℃. At reaction temperatures of 63℃, 61℃, and 59℃, the pMD19T-SsITS1 plasmid DNA from *Schisandra chinensis* began amplification from the 19th, 17th, and 17.5th cycles, respectively. No amplification was observed in the DEPC-treated water (the negative control). The reaction temperature of 61℃ resulted in the lowest number of amplification cycles and the highest amplification efficiency. Therefore, 61℃ was selected as the optimal temperature for the Proofman-LMTIA reaction of *Schisandra chinensis*.

[0086] Example 7

[0087] Single Proofman-LMTIA Specificity Test of Schisandra chinensis

[0088] Using 10 ng / μL of *Schisandra chinensis* pMD19T-SsITS1 plasmid DNA as a positive control and 10 ng / μL of *Schisandra sibirica* pMD19T-ScITS1 plasmid DNA and H2O as negative controls, the optimized Proofman-LMTIA reaction system established in Example 6 was used. The reaction was performed using a Gentier 96E fully automated medical PCR analysis system at 61°C for 40 cycles. Fluorescence signals were collected every 30 seconds for a total of 40 collections. Each reaction was performed in quadruplicate. The results were statistically analyzed and visualized using the Gentier 96E fully automated medical PCR analysis system. Specificity test results are as follows: Figure 15As shown in the figure, the exponential amplification curve corresponds to the *Schisandra chinensis* plasmid DNA, and the baseline curve corresponds to the *Schisandra sibirica* plasmid DNA and H2O. The *Schisandra chinensis* plasmid pMD19T-SsITS1 showed good amplification efficiency, while the *Schisandra sibirica* pMD19T-ScITS1 plasmid and the negative control showed no amplification. This indicates that the Proofman-LMTIA reaction system established in this embodiment of the invention has good specificity for *Schisandra chinensis* plasmid DNA.

[0089] Example 8

[0090] Single Proofman-LMTIA Sensitivity Test of Schisandra chinensis

[0091] The extracted pMD19T-SsITS1 plasmid DNA from *Schisandra chinensis* was diluted to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL, respectively, with H2O as a negative control. This was added to the optimized Proofman-LMTIA reaction system described in Example 6. Two parallel samples were set for each reaction, and the temperature was set at 61°C. Fluorescence signals were collected every 30 seconds for a total of 40 collections. Each experiment was repeated twice to determine the optimal reaction results. Sensitivity test results are as follows: Figure 16 As shown, the pMD19T-SsITS1 plasmid DNA from Schisandra chinensis at concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL all showed good amplification, while H2O did not amplify the DNA. Therefore, the sensitivity of this method is 0.1 pg / μL.

[0092] Example 9

[0093] Single Proofman-LMTIA detection limit test of Schisandra chinensis

[0094] The extracted pMD19T-SsITS1 plasmid DNA from *Schisandra chinensis* was mixed with pMD19T-ScITS1 plasmid DNA from *Schisandra sibirica* at concentrations of 0%, 0.1%, 1.0%, 5.0%, 10%, 20%, and 100%, respectively. These mixed samples were then added to the optimized Proofman-LMTIA reaction system described in Example 6. Detection was performed using a Gentier 96E fully automated medical PCR analysis system, with DEPC-treated water as a negative control. Each experiment was repeated twice. The temperature was set at 61°C, and fluorescence signals were collected every 30 seconds for a total of 40 collections. The detection limit test results are as follows: Figure 17As shown in the figure, the curves marked with 100%, 20%, 10%, 5%, 1%, and 0.1% correspond to mixed samples with pMD19T-ScITS1 plasmid DNA volumes of 100%, 20%, 10%, 5.0%, 1.0%, and 0.1%, respectively. The baseline is compared to the sample with 0% pMD19T-ScITS1 plasmid DNA volume and the negative control H2O. The results show that pMD19T-ScITS1 plasmid DNA was successfully amplified in the mixed samples with 100%, 20%, 10%, 5%, 1%, and 0.1% concentrations, while no amplification occurred in the 0% mixed sample and the negative control. Therefore, the detection limit of this method is 0.1%.

[0095] Example 10

[0096] Dual Proofman-LMTIA temperature optimization test of Schisandra chinensis

[0097] In a clean bench, add the following reaction mixture to a 100 μL PCR tube: 5 μL 2×Mix premix, 0.4 μL 2 U / μL GPV8 high-fidelity DNA polymerase, 0.16 μL 10 μmol / L primer WuWZ-F3, 0.16 μL 10 μmol / L primer WuWZ-B13, and 0.04 μL 10 μmol / L primer WuWZ-LF. The following reagents were used: 0.16 μL of 10 μmol / L WuWZ-F1, 0.04 μL of 10 μmol / L primer NWuWZ-LF, 0.3 μL of 10 μmol / L WuWZProbe, 0.3 μL of 10 μmol / L NWuWZProbe, 2 μL of *Schisandra chinensis* pMD19T-ScITS1 plasmid DNA template, and 1.44 μL of H2O. Detection was performed using a Gentier 96E fully automated medical PCR analysis system. The reaction temperatures were 58℃, 60℃, 62℃, and 64℃, with a cycle count of 40. Fluorescence signals were collected every 30 seconds for a total of 40 collections. The test sample was *Schisandra chinensis* pMD19T-ScITS1 plasmid DNA, and the negative control was H2O. Each experiment was conducted with three parallel samples, and the results were statistically analyzed and observed using the Gentier 96E fully automated medical PCR analysis system software.

[0098] Temperature optimization test results are as follows Figure 18As shown in the figure, the curves marked with temperature correspond to the pMD19T-ScITS1 plasmid DNA from *Schisandra chinensis*, while H2O serves as the negative control. At reaction temperatures of 64℃, 62℃, 60℃, and 58℃, the pMD19T-ScITS1 plasmid DNA from *Schisandra chinensis* began amplification from the 13th, 12th, 13th, and 15th cycles, respectively. The negative control H2O showed no amplification. The amplification cycle number was lower and the amplification efficiency was higher at a reaction temperature of 60℃; therefore, 60℃ was chosen as the optimal temperature for the Proofman-LMTIA reaction of *Schisandra chinensis*.

[0099] Example 11

[0100] Dual Proofman-LMTIA specificity test for Schisandra chinensis (Northern and Southern varieties)

[0101] Using 10 ng / μL of pMD19T-ScITS1 plasmid DNA from Schisandra chinensis and 10 ng / μL of pMD19T-SsITS1 plasmid DNA from Schisandra chinensis as positive controls and DEPC-treated water as a negative control, the optimized Proofman-LMTIA reaction system established in Example 10 was used for the reaction. The reaction was performed using a Gentier 96E fully automated medical PCR analysis system at a temperature of 60°C for 40 cycles. Fluorescence signals were collected every 30 seconds for a total of 40 collections. Ten parallel samples were set for each reaction. The results were statistically analyzed and visualized using the Gentier 96E fully automated medical PCR analysis system.

[0102] The results of the dual specificity test are as follows Figure 19 As shown in the figure, the exponential amplification curves correspond to the DNA of *Schisandra chinensis* plasmid and *Schisandra sibirica* plasmid, and the baseline curve corresponds to H2O. The *Schisandra chinensis* plasmid pMD19T-ScITS1 showed good amplification efficiency in the FAM channel, and the *Schisandra sibirica* plasmid pMD19T-SsITS1 showed good amplification efficiency in the JOE channel, while the negative control showed no amplification. This indicates that the dual Proofman-LMTIA reaction system established in this embodiment of the invention has good specificity for the *Schisandra chinensis* plasmid pMD19T-ScITS1 and *Schisandra sibirica* plasmid pMD19T-SsITS1.

[0103] Example 12

[0104] Dual Proofman-LMTIA Sensitivity Test of Schisandra chinensis

[0105] The extracted pMD19T-ScITS1 plasmid DNA from Schisandra chinensis was diluted to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, and 10 fg / μL, respectively. DEPC-treated water was used as a negative control. The DNA was added to the Proofman-LMTIA reaction system optimized in Example 10. Three parallel samples were set for each reaction. The temperature was set at 60°C. Fluorescence signals were collected every 30 seconds for a total of 40 collections.

[0106] Sensitivity test results as follows Figure 20 As shown, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, and 10 fg / μL of Schisandra chinensis pMD19T-ScITS1 plasmid DNA were all well amplified, while H2O did not amplify the DNA. Therefore, the sensitivity of this method is 10 fg / μL.

[0107] Example 13

[0108] Dual Proofman-LMTIA detection limit test of Schisandra chinensis

[0109] The extracted pMD19T-ScITS1 plasmid DNA from *Schisandra chinensis* was mixed with pMD19T-SsITS1 plasmid DNA from *Schisandra spp.* at concentrations of 0%, 0.1%, 1.0%, 5.0%, 10%, 20%, and 100%, respectively. These mixed samples were then added to the optimized Proofman-LMTIA reaction system described in Example 10. Detection was performed using a Gentier 96E fully automated medical PCR analysis system, with H2O as a negative control. Each experiment was repeated three times. The temperature was set at 60°C, and fluorescence signals were collected every 30 seconds for a total of 40 collections.

[0110] Detection limit test results as follows Figure 21As shown in the figure, the curves marked with 100%, 20%, 10%, 5%, 1%, and 0.1% correspond to samples with pMD19T-ScITS1 plasmid DNA volume fractions of 100%, 20%, 10%, 5.0%, 1.0%, and 0.1%, respectively. The baseline corresponds to samples with pMD19T-SsITS1 plasmid DNA volume fractions of 0.1% and 0%, and the negative control DEPC-treated water. The results show that pMD19T-ScITS1 plasmid DNA was successfully amplified in the mixed samples with 100%, 20%, 10%, 5%, and 1% concentrations, while no amplification occurred in the mixed samples with 0.1% and 0% concentrations, or the negative control. Therefore, the detection limit of this method is 1%.

[0111] Example 14

[0112] Dual Proofman-LMTIA temperature optimization test of Schisandra chinensis

[0113] Temperature optimization was performed using the Proofman-LMTIA method. In a clean bench, the following reaction mixture was added to a 100 μL PCR tube: 5 μL 2×Mix premix, 0.4 μL 2 U / μL GPV8 high-fidelity DNA polymerase, 0.16 μL 10 μmol / L WuWZ-F3 primer, 0.16 μL 10 μmol / L WuWZ-B13 primer, and 0.04 μL 10 μmol / L WuWZ-LF primer. 0.16 μL of 10 μmol / L WuWZ-F1, 0.04 μL of 10 μmol / L NWuWZ-LF, 0.3 μL of 10 μmol / L WuWZProbe, 0.3 μL of 10 μmol / L NWuWZProbe, 2 μL of *Schisandra chinensis* pMD19T-SsITS1 plasmid DNA template, and 1.44 μL of DEPC-treated water were used for detection. A Gentier 96E fully automated medical PCR analysis system was used for detection. The reaction temperatures were 58℃, 60℃, 62℃, and 64℃, with a cycle count of 40. Fluorescence signals were collected every 30 seconds for a total of 40 collections. The test sample was *Schisandra chinensis* pMD19T-SsITS1 plasmid DNA, and the negative control was H2O. Each experiment was conducted with two parallel samples, and the results were statistically analyzed and observed using the Gentier 96E fully automated medical PCR analysis system software.

[0114] Temperature optimization test results are as follows Figure 22As shown in the figure, the curves marked with temperature correspond to the pMD19T-SsITS1 plasmid DNA from *Schisandra chinensis*, with H2O serving as the negative control. At reaction temperatures of 64℃, 62℃, 60℃, and 58℃, the pMD19T-ScITS1 plasmid DNA from *Schisandra chinensis* amplified starting from the 14th, 14th, 12th, and 14th cycles, respectively. The negative control, DEPC-treated water, showed no amplification. The reaction temperature of 60℃ resulted in the fewest amplification cycles and the highest amplification efficiency; therefore, 60℃ was selected as the optimal reaction temperature for the dual Proofman-LMTIA reaction of *Schisandra chinensis*.

[0115] Example 15

[0116] Dual Proofman-LMTIA Sensitivity Test of Schisandra chinensis

[0117] The extracted pMD19T-SsITS1 plasmid DNA from Schisandra chinensis was diluted to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL, respectively. H2O was used as a negative control. The DNA was added to the Proofman-LMTIA reaction system optimized in Example 14. Three parallel samples were set for each reaction. The temperature was set at 60°C. Fluorescence signals were collected every 30 seconds for a total of 40 collections.

[0118] Sensitivity test results as follows Figure 23 As shown, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL of *Schisandra chinensis* pMD19T-SsITS1 plasmid DNA were all well amplified, while no amplification was observed with H2O. Therefore, the sensitivity of this method is 1 fg / μL.

[0119] Example 16

[0120] Dual Proofman-LMTIA detection limit test of Schisandra chinensis

[0121] The extracted pMD19T-SsITS1 plasmid DNA from *Schisandra chinensis* was mixed with pMD19T-ScITS1 plasmid DNA from *Schisandra sibirica* at concentrations of 0%, 0.1%, 1.0%, 5.0%, 10%, 20%, and 100%, respectively. These mixed samples were then added to the optimized Proofman-LMTIA reaction system described in Example 14. Detection was performed using a Gentier 96E fully automated medical PCR analysis system, with DEPC-treated water as a negative control. Each experiment was repeated twice. The temperature was set at 60°C, and fluorescence signals were acquired every 30 seconds for a total of 40 acquisitions.

[0122] Detection limit test results as follows Figure 24 As shown in the figure, the curves marked with 100%, 20%, 10%, 5%, 1%, and 0.1% correspond to mixed samples with pMD19T-SsITS1 plasmid DNA volumes of 100%, 20%, 10%, 5.0%, 1.0%, and 0.1%, respectively. The baseline corresponds to the sample with 0% pMD19T-SsITS1 plasmid DNA volume and the negative control H2O. The results show that pMD19T-SsITS1 plasmid DNA was successfully amplified in the mixed samples with 100%, 20%, 10%, 5%, 1%, and 0.1% concentrations, while no amplification occurred in the 0% mixed sample and the negative control. Therefore, the detection limit of this method is 0.1%.

[0123] Example 17

[0124] Dual Proofman-LMTIA testing of Schisandra chinensis (Southern and Northern varieties) processed products

[0125] Seven samples of Schisandra chinensis (Northern) and one sample of Schisandra chinensis (Southern) were taken from the market. Genomic DNA was extracted according to the method in Example 1, and the results were analyzed according to the method in Example 10. The reaction was performed using a Gentier 96E fully automated medical PCR analysis system at 60°C for 40 cycles. Fluorescence signals were collected every 30 seconds for a total of 40 collections. Each reaction was performed in triplicate. The results were statistically analyzed and visualized using the Gentier 96E fully automated medical PCR analysis system. The results are as follows: Figure 25 As shown, the genomic DNA of 7 samples of Schisandra chinensis from the north and 1 sample of Schisandra chinensis from the south was successfully amplified, while the negative control H2O did not produce amplification, indicating that this method can be used for the rapid detection of both Schisandra chinensis from the north and south.

[0126] Example 18

[0127] Dual Proofman-LMTIA detection of medicines containing Schisandra chinensis and Schisandra chinensis.

[0128] Five commercially available medicines containing Schisandra chinensis were collected. Genomic DNA was extracted according to the method in Example 1, and the samples were tested according to the method in Example 10. The reaction was performed using a Gentier 96E fully automated medical PCR analysis system at 60°C for 40 cycles. Fluorescence signals were collected every 30 seconds for a total of 40 collections. Each reaction was performed in triplicate. The results were statistically analyzed and visualized using the Gentier 96E fully automated medical PCR analysis system. The test results are as follows: Figure 26 As shown, the genomic DNA of lily bulb, jujube seed, polygala root, schisandra fruit, and ophiopogon root capsules, brain-nourishing capsules, brain-strengthening tablets, Shengmai drink, and strong brain-clearing tablets all showed good amplification, while the negative control H2O showed no amplification. This indicates that this method can be used for the rapid detection of drugs containing schisandra fruit.

[0129] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An LMTIA primer set and Proofman probe for dual detection of Schisandra chinensis and Schisandra chinensis, characterized in that, The LMTIA primer set and Proofman probe were designed with ITS1 as the target sequence of Schisandra chinensis and Schisandra sibirica. The LMTIA primer set includes: WuWZ-F1 as shown in sequence SEQ ID NO 1, WuWZ-B13 as shown in sequence SEQ ID NO 2, WuWZ-F3 as shown in sequence SEQ ID NO 3, WuWZ-LF as shown in sequence SEQ ID NO 4, and NWuWZ-LF as shown in sequence SEQ ID NO 5; The Proofman probes include WuWZProbe as shown in sequence SEQ ID NO 6 and NWuWZProbe as shown in sequence SEQ ID NO 7.

2. The LMTIA primer set and Proofman probe for dual detection of Schisandra chinensis and Schisandra chinensis according to claim 1, characterized in that, The 5' end of the WuWZProbe is labeled with a BHQ2 quenching group, and the 3' end is labeled with a FAM fluorescent group; the 5' end of the NWuWZProbe is labeled with a BHQ2 quenching group, and the 3' end is labeled with a JOE fluorescent group.

3. A reagent kit for detecting the authenticity of Schisandra chinensis (Southern and Northern varieties), characterized in that, It includes the LMTIA primer set and Proofman probe as described in claim 1.

4. The application of the LMTIA primer set and Proofman probe for dual detection of Schisandra chinensis and Schisandra spp. as described in claim 1, or the kit for authenticity detection of Schisandra chinensis and Schisandra spp. as described in claim 3, in the detection of seeds, seedlings, vines, processed slices, food and pharmaceuticals containing Schisandra chinensis and Schisandra spp.

5. A method for detecting Schisandra chinensis from both North and South China based on dual Proofman-LMTIA, characterized in that, Includes the following steps: Take DNA from the sample to be tested, prepare a Proofman-LMTIA reaction system, and observe the fluorescence signal or amplification curve after isothermal amplification to detect whether there is Schisandra chinensis and / or Schisandra spp. in the sample to be tested; the Proofman-LMTIA reaction system includes the LMTIA primer set and Proofman probe as described in claim 1.

6. The detection method of Schisandra chinensis and Schisandra chinensis based on dual Proofman-LMTIA according to claim 5, characterized in that, The molar ratio of WuWZ-F1:WuWZ-B13:WuWZ-F3:WuWZ-LF:NWuWZ-LF:WuWZ-Probe:NWuWZ-Probe in the Proofman-LMTIA reaction system is 8:8:8:8:2:2:15:

15.

7. The detection method for Schisandra chinensis and Schisandra chinensis based on dual Proofman-LMTIA according to claim 5, characterized in that, The isothermal amplification procedure is as follows: 60℃, 40 cycles, fluorescence signal is collected every 30 seconds, for a total of 40 fluorescence signals.

8. The detection method for Schisandra chinensis and Schisandra chinensis based on dual Proofman-LMTIA according to claim 5, characterized in that, The Proofman-LMTIA reaction system has a volume of 10 μL and contains 5 μL of 2×Mix premix, 0.4 μL of ultra-fidelity DNA polymerase, 0.16 μL of WuWZ-F1, 0.16 μL of WuWZ-B13, 0.16 μL of WuWZ-F3, 0.04 μL of WuWZ-LF, 0.04 μL of NWuWZ-LF, 0.3 μL of WuWZProbe, 0.3 μL of NWuWZProbe, 2 μL of genomic DNA template, and 1.44 μL of water.

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