CN109385487B - Recombinase-mediated amplification isothermal detection method and kit for American ginseng as Chinese medicinal herb - Google Patents

Abstract

Translated fromChinese

本发明提供一种供用于检测中药中西洋参物种（Panax quinquefolius）的重组酶介导扩增（Recombinase‑aid Amplification，RAA）恒温检测方法和检测试剂盒。检测试剂盒包括一条正向引物SEQ ID NO.1、一条反向引物SEQ ID NO.2、一条特异性荧光探针SEQ ID NO.3、反应液、重组聚合酶和对照品。本发明的试剂盒特异性强；检测灵敏度高，可达到0.10fg/μL；准确度高、可靠；操作简便快捷，适合现场检测，具有广泛的应用场景。

The present invention provides a recombinase-aid Amplification (RAA) constant temperature detection method and a detection kit for detectingPanax quinquefolius in traditional Chinese medicine. The detection kit includes a forward primer SEQ ID NO.1, a reverse primer SEQ ID NO.2, a specific fluorescent probe SEQ ID NO.3, a reaction solution, a recombinant polymerase and a reference substance. The kit of the invention has strong specificity; high detection sensitivity, which can reach 0.10 fg/μL; high accuracy and reliability; simple and fast operation, suitable for on-site detection, and has a wide range of application scenarios.

Description

Recombinase-mediated amplification isothermal detection method and kit for American ginseng as Chinese medicinal herb

Technical Field

The invention belongs to the field of precious Chinese herbal medicine detection and the technical field of molecular biology, and particularly relates to a method for identifying whether American ginseng (Panax quinquefolius) components are contained in a traditional Chinese medicine by adopting a Recombinase-aid Amplification (RAA) constant-temperature detection technology and combining a specific primer and a fluorescent probe.

Background

Since 2017, the intensive new administration of traditional Chinese medicine, especially the implementation of the traditional Chinese medicine law and the release of the poverty alleviation action plan (2017 Busy 2020) of the traditional Chinese medicine industry bring new opportunities for the development of the traditional Chinese medicine industry. Under the background, the Chinese medicinal material supply scale in China continues to be enlarged, circulation link resources are optimized and promoted, intensive production place processing modes are highlighted, an 'internet plus' novel trade mode is started, the Chinese medicinal material circulation market accelerates transformation and upgrading, and Chinese medicinal material import and export trades are increased. 9.10 million tons of Chinese medicinal materials are imported in China in the last year, the year-by-year ratio is increased by 13.62%, the average import price is $ 2.87/kg, the year-by-year ratio is increased by 14.15%, the total import amount of the Chinese medicinal materials is $ 2.61 billion, the year-by-year ratio is increased by 29.69%, and the year-by-year ratio is much higher than the year-by-year ratio increase of the import amount of goods in China by 18.7%. The main varieties imported in China comprise longan, American ginseng, pilose antler, saffron, frankincense, myrrh, dragon's blood and the like.

American ginseng is the only variety with the import sum higher than the export sum in the top entrance and exit ten varieties. People who have long-term operation of American ginseng know that imported American ginseng has a lot of greasy cats mixed with fish and dragon. American ginseng is imported very rarely, if at all, unlike Canada. According to the analysis and report of each traditional Chinese medicine industry, about 1500 tons of American ginseng are imported every year, but less than 800 tons or even less are imported from customs officially according to statistics, and many American ginseng are imported from hong Kong without influencing the import quantity. At present, a large number of false mixing and false making behaviors exist in American ginseng in China, the quality and the medication safety of traditional Chinese medicines are seriously influenced, and how to simply, quickly and accurately detect whether the nominal American ginseng traditional Chinese medicine is real American ginseng becomes a problem to be solved urgently.

The Recombinase-aid Amplification (RAA) technique is also a method by which nucleic acids can be rapidly amplified at a constant temperature. Unlike RPA, RAA amplification uses a recombinase obtained from bacteria or fungi, which binds tightly to the primer DNA at a constant temperature of 37 ℃ to form an aggregate of the enzyme and the primer, when the primer searches for a sequence on the template DNA that is completely complementary to the primer, the template DNA is melted with the help of single-stranded DNA binding protein (SSB), and a new complementary strand of DNA is formed under the action of DNA polymerase, and the reaction product is exponentially increased, and usually an amplified fragment that can be detected by agarose gel electrophoresis can be obtained within 1 hour. The fluorescent group is added into the RAA reaction system, the whole RAA amplification process is monitored in real time by utilizing the accumulation of fluorescent signals, and the quantitative and qualitative analysis of the initial template can be realized within 20 minutes. The whole reaction is simple and quick, and high-temperature circulation is not needed, so the method is particularly suitable for non-laboratory detection places with a large number of samples, and is suitable for the field of rapid identification and detection of the truth of precious Chinese herbal medicines.

Disclosure of Invention

The invention aims to provide a recombinase-mediated amplification isothermal detection method and a kit for American ginseng (Panax quinquefolius) which is a precious Chinese herbal medicine.

In order to achieve the purpose, the invention adopts the following technical scheme:

a detection kit for nucleic acid of American ginseng, a precious Chinese herbal medicine, comprises: the primer comprises an American ginseng forward primer, a reverse primer and a specific fluorescent probe, wherein the nucleotide sequence of the American ginseng forward primer is shown as SEQ ID No.1, the nucleotide sequence of the American ginseng reverse primer is shown as SEQ ID No.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID No.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group.

In some embodiments, the fluorescent reporter group of the specific fluorescent probe is selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC Red460, and the fluorescence quenching gene is selected from one of BHQ1, BHQ2, BHQ3, DABCY1, or TAMRA.

In some embodiments, the nucleic acid detection kit further comprises a primer mixture, a specific fluorescent probe, an A Buffer, a B Buffer, a RAA dry powder reagent, an American ginseng standard and ddH₂At least one of O.

In some embodiments, the kit, wherein the said A Buffer is 20% PEG; b Buffer is 280mM MgAc.

In some embodiments, the kit, wherein the composition of the RAA dry powder reagent is as follows: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ L Rad51, 30ng/μ L Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, Exo exonuclease.

In some embodiments, in the nucleic acid detection kit, the american ginseng standard is a positive plasmid containing a partial sequence of the entire genome of american ginseng.

In some embodiments, the kit, the positive plasmid containing the whole genome partial sequence of panax quinquefolium is shown in SEQ ID No. 4.

The invention also provides an RAA constant temperature detection method of American ginseng, which comprises the steps of extracting DNA of a sample to be detected, taking the DNA of the sample to be detected as a template, and performing amplification on a forward primer, a reverse primer, a specific fluorescent probe, an RAA dry powder reagent, an A Buffer, a B Buffer and a ddH of the American ginseng₂Carrying out real-time fluorescence RAA reaction in the presence of O, and analyzing a sample to be detected according to a real-time fluorescence RAA amplification curve; wherein said American ginseng forward primer nucleosideThe sequence is shown as SEQ ID NO.1, the American ginseng reverse primer nucleotide sequence is shown as SEQ ID NO.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID NO.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group.

In some embodiments, the real-time fluorescent RAA reaction program is: 39 ℃, 40s, one cycle; at 39 ℃ for 30s for one cycle, 20min for 40 cycles.

According to the detection method, after the real-time fluorescence RAA reaction is required to be finished, the to-be-detected sample is analyzed according to the amplification curve of the real-time fluorescence RAA by using the analysis software of the real-time fluorescence RAA instrument. Preferably, the FAM channel fluorescence curve of the sample to be tested is S-shaped and the CT value is less than or equal to 35, and the sample to be tested is judged to be the American ginseng positive result; and when the curve of the sample to be detected is not S-shaped or the CT value is more than 35, judging the result as the American ginseng negative result.

Has the advantages that:

1. fast and efficient: the whole amplification can be completed within 20-30min, and the amplification yield can reach 10⁹-10¹⁰A copy;

2. the operation is simple: no special reagent is needed, complicated steps such as deformation of double-stranded DNA and the like are not needed in advance, only a constant-temperature fluorometer is needed, and the conditions are mild;

3. high specificity: the invention does not amplify DNA of other components of American ginseng, such as ginseng (Panax ginseng C.A. Meyer), notoginseng (Burk.) F.H.Chen, etc.

4. High sensitivity: the detection limit of the invention can reach 0.10 fg/muL reaction.

5. The identification is simple: and the amplification result is directly judged according to the real-time fluorescence data, electrophoresis detection is not needed, and the method is suitable for field detection.

Drawings

FIG. 1 is a graph showing RAA amplification curves of 3 pairs of primers involved in the present invention.

FIG. 2 is a sensitivity test chart of the RAA detection method on American ginseng, and the amplification results of positive standard substances of 100fg/μ L, 10.0fg/μ L, 1.00fg/μ L, 0.10fg/μ L, 0.01fg/μ L and 1.00ag/μ L are sequentially performed from left to right.

FIG. 3 is a diagram of the specificity experiment of the RAA detection method on American ginseng.

Detailed description of the invention

The present invention is further illustrated by the following specific examples, but is not limited thereto.

Example 1: primer and probe design screening

The invention searches the whole gene sequence of the American ginseng in a Genebank database, and compares a plurality of sequences by using DNAMAN 6.0 software to find out a conservative section. 3 sets of primers and probes were designed in conserved regions and BLAST alignments were performed in the NCBI database, with the sequences of the primers and probes as shown in Table 1.

For the positive plasmid of the gene sequence of the American ginseng conserved region, the base sequence of the plasmid is represented by SEQ ID NO.4,

ATGGCCCATCACGCCCAACAGGTAAAAGGGGTGCATGGCATGCATGGA AATCTAGAAAGGTACCATCGCGTGAGCTAAGCCCCATCGTGTTGAGCAACC AAAATCCAACGCAAGGAGATAAATCTTGAGTCGAGGTTAACACGACCTTTT TTGTTTCATGTGTGAGGCGAAGAGGTCCACGGCCCGTAGCACGCCGTGGC AGGGCCCAACACGCCCAGAGGCAGCACCACGCACACGCAAACAGGCAAC GCCAACCAGGACCATCGAATGAAATGGCGGCAAACATGAAAAAAGCTTGC ACTACACCATTTCCTCGAGCTA(SEQ ID NO.4)

the reagents for amplification were as follows:

comprises primer mixed liquor, a specific fluorescent probe, an A Buffer, a B Buffer, an RAA dry powder reagent, an American ginseng standard substance and ddH₂And O. The ground A Buffer is 20% PEG; b Buffer is 280mM MgAc.

The RAA dry powder reagent comprises the following components: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ L Rad51, 30ng/μ L Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, and Exo exonuclease.

The real-time fluorescent RAA reaction program is as follows: 39 ℃, 40s, one cycle; at 39 ℃ for 30s for one cycle, 20min for 40 cycles.

According to the detection method, after the real-time fluorescence RAA reaction is required to be finished, the to-be-detected sample is analyzed according to the amplification curve of the real-time fluorescence RAA by using the analysis software of the real-time fluorescence RAA instrument.

The positive sample amplification curve is shown in FIG. 1.

Table 1 primer and probe sequences:

as can be seen from the results in FIG. 1, the amplification curves for the first set of primers and probes are most typical, with distinct exponential and plateau phases, with higher fluorescence intensity (ordinate values), and smaller CT values (abscissa corresponding to the intersection of the curve with the threshold line) and the results are analyzed in Table 2. The rise height of other primer probe curves is lower, the CT value is larger, and the plateau period is not obvious; or no amplification occurs and missed detection occurs. The first group of primers and the target products of the probes are higher in replication speed, more in quantity and higher in amplification reaction efficiency.

TABLE 2 analysis of primer Probe screening results

Group/result	CT value	Intensity of fluorescence
			First group	16.97	690000
Second group	35.21	500000
			Third group	40.21	450000

Example 2: kit assembling method

American ginseng of the kit

The nucleic acid detection kit also comprises a primer mixed solution, a specific fluorescent probe, an A Buffer, a B Buffer, an RAA dry powder reagent, an American ginseng standard substance and ddH₂And O. The kit provided by the invention, wherein the GeA Buffer is 20% PEG; b Buffer is 280mM MgAc. The kit of the invention, wherein the RAA dry powder reagent comprises the following components: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ L Rad51, 30ng/μ L Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, Exo exonuclease.

In the primer mixture, the base sequence of the forward primer is shown as SEQ ID NO.1, the base sequence of the reverse primer is shown as SEQ ID NO.2, and the molar ratio of the forward primer to the reverse primer is SEQ ID NO. 1: SEQ ID NO.2 is 1: 1. The base sequence of the specific probe of American ginseng provided by the invention is shown in SEQ ID NO.3, the 5 'end of the probe is marked with FAM fluorescent reporter group, and the 3' end of the probe is marked with BHQ1 fluorescent quenching group. The American ginseng standard product provided by the invention comprises a positive plasmid of an American ginseng conserved region gene sequence, and the base sequence of the plasmid is shown in SEQ ID NO. 4.

Example 3: application method of kit

1. Extraction of nucleic acids from Positive samples

1.1, nucleic acid extraction: extracting plant genome DNA nucleic acid by CTAB improvement method.

Adding the same tissue powder into 700 mu L of 2 xCTAB extraction buffer solution preheated in a 65 ℃ water bath, gently stirring, preserving the temperature in a 1.5ml sterilized centrifuge tube in a 65 ℃ water bath or a thermostat for 40min, and gently shaking every 10 min;

② after cooling for 2min, adding equal volume of chloroform: mixing isoamyl alcohol (24: 1) uniformly, centrifuging for 10min at L0000r/min, taking supernatant, and transferring into an isopropanol centrifuge tube with 600 mu L;

thirdly, the mixture is shaken for 30s to obtain visible DNA floccules;

fourthly, after 1min of centrifugation at 0000r/min, immediately pouring out the liquid to keep white DNA precipitate, standing upside down for 60s, adding 720 mu L of 75% ethanol and 80 mu L of 5M sodium acetate, and standing at room temperature for 30 min;

fifthly, centrifuging at L0000r/min for 1min, pouring out liquid, adding 800 microliter of 75% ethanol, standing for 30min, centrifuging at L0000r/min for 30s, and pouring out liquid;

sixthly, after a plurality of minutes, adding 50 mu L of 0.5 XTE buffer solution into the dried DNA, and placing the DNA in a constant temperature cabinet at 37 ℃ for about 15 hours;

seventhly, preserving at-20 ℃ for later use.

2. Configuration of RAA reaction system: one RAA reaction dry powder tube (reagent as described in example 2) was used for each test sample, and the reaction components and the added volume in each RAA reaction dry powder tube are shown in Table 3.

Table 3:

a Buffer is 20% PEG; b Buffer is 280mM MgAc.

3. Placing the RAA reaction tube with the prepared reaction system in an ABI7500 amplification instrument, and carrying out RAA amplification according to the following procedures: 39 ℃, 40s, one cycle; at 39 ℃ for 30s for one cycle, 20min for 40 cycles. Fluorescence of FAM channels was collected for each cycle.

4. And after the amplification is finished, judging the positive or negative result of the American ginseng according to the fluorescence curve judgment and the CT value.

And (4) judging the result: the fluorescence curve of the FAM channel is S-shaped, the CT value is less than or equal to 35, and the result is judged to be the positive result of the American ginseng; and when the curve of the sample to be detected is not in an S shape or the CT value is more than 35, judging the American ginseng negative result (the judgment standard is obtained according to theembodiments 1, 2, 4, 5 and 6).

Example 4: evaluation of RAA detection kit of the invention in practical application

Blind experiments (reagent of example 2 and detection method of example 3) were performed using the kit of the present invention to detect 34 precious Chinese herbal medicines; experimental results show that the American ginseng can be distinguished by the first primer pair, and the positive coincidence rate with the nested PCR is high. In 34 parts, nested PCR (same primers and actual proportion according to traditional PCR amplification) has 18 parts of positive results and 16 parts of negative results (34 is negative), the result detected by the RAA method has 18 parts of positive results and 15 parts of negative results, one positive result is different (34 is positive), the DNA of the sample is subjected to PCR amplification and sequencing, the sequencing result shows that the sample is positive, the RAA detection reagent has higher accuracy, and the results are shown in Table 4. That is, the RAA test has 19 positives and 15 negatives, while the traditional nested PCR has only 18 positive results and 16 negative results.

Similarly, the RAA assay using the second and third sets of primers of the invention found only 12 positive and 22 negative results (second set); in the third group, only 10 were positive and 24 were negative. (the specific experimental process is not shown).

Table 4: 34 unknown Blind sample test results (RAA test results)

Example 5: sensitivity test of the kit of the invention

The American ginseng standard plasmid provided by the kit in theembodiment 2 of the invention is used for extracting positive plasmids, measuring the concentration of the positive plasmids by using NanoDrop, and diluting the positive plasmids to six concentration gradients of 100 fg/muL, 10.0 fg/muL, 1.00 fg/muL, 0.10 fg/muL, 0.01 fg/muL and 1.00 ag/muL respectively for carrying out sensitivity test.

The detection results are shown in figure 2, and are the amplification results of positive standard substances of 100 fg/muL, 10.0 fg/muL, 1.00 fg/muL, 0.10 fg/muL, 0.01 fg/muL and 1.00 ag/muL from left to right in sequence, so that the RAA fluorescence amplification reagent and the detection sensitivity can reach 0.10 fg/muL, the accuracy is superior to that of the common PCR detection method, and the RAA constant temperature detection kit and the detection method have high sensitivity to the diagnosis of American ginseng. The same primers are adopted, the traditional PCR is used for amplification, and the sensitivity can only detect the amount of 1.00 ag/mu L (the specific experimental process is not shown). The RAA of the invention has a 10-fold higher sensitivity than conventional methods. While the sensitivity of the other second and third groups was very low, the lowest detection threshold of the second group was 10.0 fg/. mu.L and the lowest detection threshold of the third group was 100 fg/. mu.L.

Test example 6: specificity test of the kit of the present invention

In order to detect the specificity of the kit, 20 samples of ginseng (Panax ginseng C.A.Mey), notoginseng (Panax notogeng (Burk.) F.H.Chen) and the like which are not American ginseng are respectively detected by the detection method in example 3, and the detection conditions of the kit on American ginseng (Panax quinquefolius) and other common components of Chinese herbal medicines in Guijun are analyzed.

The detection result shows that: normal amplification occurred only in the American Ginseng sample, negative control (ddH)₂O) and ginseng (Panax ginseng c.a.mey), notoginseng (Panax notoginseng (Burk.) f.h.chen), etc. all other 20 samples were not amplified (fig. 3 shows the amplification curves of Panax quinquefolius and ginseng). The results show that the RAA constant temperature detection kit can specifically amplify the target sequence in the American ginseng, and does not have cross reaction with other precious Chinese herbal medicine nucleic acids. The method and the kit have good specificity and do not generate false negative.

Meanwhile, 2-3 pairs of primers designed by the invention are used for carrying out the same specificity experiment, and the primers can not distinguish different samples well in a specific way, so that the specificity is not good (the specific experimental data is slight).

The invention shown and described herein may be practiced in the absence of any element or elements, limitation or limitations, which is specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It should therefore be understood that although the present invention has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The contents of the articles, patents, patent applications, and all other documents and electronically available information described or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.

Sequence listing

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Claims (4)

1. A RAA detection method of traditional Chinese medicine American ginseng (Panax quinquefolius) comprises an American ginseng forward primer, an American ginseng reverse primer and a specific fluorescent probe, and is characterized in that the nucleotide sequence of the American ginseng forward primer is shown as SEQ ID NO. 1; the nucleotide sequence of the American ginseng reverse primer is shown as SEQ ID NO. 2; the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID NO.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group;

the detection method comprises the following steps:

(1) extracting genome DNA from a traditional Chinese medicine sample to be detected as a template; carrying out RAA amplification on a template by using the American ginseng forward primer, the American ginseng reverse primer and the specific fluorescent probe; the reaction temperature of RAA amplification is 30-42 ℃, and the reaction time is 5-30 min; the reaction procedure for the RAA amplification was: 39 ℃, 40s, one cycle; at 39 ℃, 30s of one cycle, 20min and 40 cycles;

(2) the judgment is carried out according to the following method:

determining whether the traditional Chinese medicine sample to be detected contains American ginseng or not according to the fluorescence curve of the fluorescence channel by the following method: if the fluorescence channel fluorescence curve is in an S shape and the CT value is less than or equal to 35, the Chinese medicine sample to be detected contains or is candidate to contain American ginseng; when the fluorescence curve is not in an S shape or the CT value is more than 35, the Chinese medicine sample to be detected does not contain American ginseng.

2. The detection kit for detecting or assisting in detecting the traditional Chinese medicine American ginseng is characterized by comprising a primer mixed solution and a specific fluorescent probe, wherein the primer mixed solution comprises an American ginseng forward primer and a reverse primer, and the nucleotide sequence of the American ginseng forward primer is shown as SEQ ID No. 1; the nucleotide sequence of the American ginseng reverse primer is shown as SEQ ID NO. 2; the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID NO.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group;

the kit also comprises an American ginseng standard substance, an A Buffer, a B Buffer, a RAA dry powder reagent and ddH 2O;

the A Buffer is 20% PEG; the B Buffer is 280mM MgAc;

the components of the RAA dry powder reagent are as follows: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein or 30ng/μ L Rad51, 30ng/μ L Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, and Exo exonuclease.

3. The detection method of claim 1 or the kit of claim 2, wherein the fluorescent reporter group is selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC Red 460; the fluorescence quenching gene is selected from one of BHQ1, BHQ2, BHQ3, Dabcy1 or TAMRA.

4. The kit of claim 2, wherein the American ginseng standard is a positive plasmid containing a partial sequence of the whole genome of American ginseng; the whole genome partial sequence of the American ginseng is shown in SEQ ID NO. 4.

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