CN112725511A

CN112725511A - Micro-drop digital PCR (polymerase chain reaction) primer, probe, kit and method for quantitatively detecting dendrobium officinale

Info

Publication number: CN112725511A
Application number: CN202110195781.4A
Authority: CN
Inventors: 张卫东; 李伟刚; 林伟; 闫邦奇; 水克娟; 石振
Original assignee: Gongbei Customs Technology Center
Current assignee: Gongbei Customs Technology Center
Priority date: 2021-02-22
Filing date: 2021-02-22
Publication date: 2021-04-30

Abstract

The invention belongs to the field of molecular biological detection, and discloses a micro-drop digital PCR primer, a probe, a kit and a method for quantitatively detecting dendrobium officinale. The nucleotide sequences of the primers are shown as 5'-ACACTTCTTGGAAACGCCGTC-3' (SEQ ID NO.1) and 5'-CGTACAGAATACGATATCCCTC-3' (SEQ ID NO.2) below. The nucleotide sequence of the probe is shown as follows: 5'-CCTCCCTATCGTGGGACAGACAG-3' (SEQ ID NO. 3). The specific primers and the probes can be used for quantitatively detecting the dendrobium officinale, and have good specificity and sensitivity.

Description

Micro-drop digital PCR (polymerase chain reaction) primer, probe, kit and method for quantitatively detecting dendrobium officinale

Technical Field

The invention belongs to the field of molecular biological detection, and particularly relates to a micro-drop digital PCR primer, a probe, a kit and a method for quantitatively detecting dendrobium officinale.

Background

Dendrobium officinale is an orchid family herbaceous plant, has upright stem, cylindrical shape, length of 9-35cm, thickness of 2-4mm, no branch, multiple segments, internode length of 1-3-1.7cm, and 3-5 leaves which are often grown above the middle part; the second row of leaves is made of paper, the long round shape is in a needle shape, the length is 3-4cm, the width is 9-11mm, the tip is blunt, the base part extends downwards to form a sheath for holding the stem, and the edge and the middle rib are always purplish; the leaf sheath is usually purple-spotted, and when old, the upper edge of the leaf sheath loosens and opens away from the stem, and 1 annular iron blue gap is left between the leaf sheath and the node. The dendrobium officinale has the effect of promoting the production of body fluid, mainly shows the effects of promoting glandular secretion and organ movement, reducing the value of diabetes blood sugar induced by streptozotocin, promoting the phagocytic function of macrophages of tumor-bearing animals, enhancing the proliferation and differentiation of T lymphocytes and the activity of NK cells, and obviously improving the value of serum hemolysin of the tumor-bearing animals, and shows that the dendrobium officinale has certain improvement effect on nonspecific immunity function, or specific cellular immunity and humoral immunity function.

Because the traditional Chinese medicinal material is valuable, lawless persons often pretend to be the best of other raw materials, or other raw materials are doped in the dendrobium officinale. In order to meet the requirements of inspection and supervision work and counterfeit prevention and law enforcement of related departments, an accurate, convenient, sensitive and quantifiable dendrobium officinale detection method is hopefully provided.

The currently used molecular biological detection methods for dendrobium officinale are few, mainly conventional PCR or fluorescent quantitative PCR, the conventional PCR detection is complicated and inconvenient, and a standard curve used by the fluorescent quantitative PCR influences a measurement result, so that the methods cannot meet the actual detection requirement.

The digital PCR is used as a new technology for nucleic acid quantification, essentially divides a traditional PCR reaction into tens of thousands of PCR reactions, and independently amplifies and detects target sequences in the tens of thousands of reaction units, thereby reducing the interference of background sequences and inhibitors on the PCR reactions and greatly improving the detection sensitivity. In which, ddPCR (micro-droplet digital PCR) can carry out accurate quantitative detection without depending on Ct value and standard curve, and solves the problem that the standard curve used by fluorescence quantitative PCR affects the measurement result. Therefore, the invention hopes to develop a detection method which can be better used for detecting the dendrobium officinale.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the micro-drop digital PCR primer, the probe, the kit and the method for quantitatively detecting the dendrobium officinale, which can be used for quantitatively detecting the dendrobium officinale and have good specificity and sensitivity.

A micro-drop digital PCR primer for quantitatively detecting dendrobium officinale comprises a specific primer for amplifying an ITS (TPSH-ITS) gene of the dendrobium officinale, wherein the nucleotide sequence of the specific primer is as follows:

5’-ACACTTCTTGGAAACGCCGTC-3’(SEQ ID NO.1)，

5’-CGTACAGAATACGATATCCCTC-3’(SEQ ID NO.2)。

the invention designs specific primers and probes based on ITS (internal transcribed spacer) sequences of dendrobium officinale, and the length of a fragment obtained by amplification of the primers is 98 bp.

A micro-drop digital PCR probe for quantitatively detecting dendrobium officinale comprises a specific probe targeting an ITS gene of the dendrobium officinale, wherein the nucleotide sequence of the specific probe is as follows:

5’-CCTCCCTATCGTGGGACAGACAG-3’(SEQ ID NO.3)。

preferably, the probe is labeled with a fluorescent group and a quenching group.

The fluorescent group is preferably one of FAM, TET, HEX, CY3, JOE, VIC, ROX or CY 5; the quenching group is preferably one of BHQ1, BHQ2, TAMRA, DABCYL, MGB or Eclipse.

A detection tool comprises the primer for detecting the dendrobium officinale and/or the probe for detecting the dendrobium officinale.

Preferably, the detection tool further comprises a detection material of an internal reference gene. More preferably, the reference gene is ZW-5.8S gene.

Further preferably, the detection material for the reference gene comprises a primer and a probe for detecting the reference gene;

the nucleotide sequence of the primer for detecting the reference gene is as follows:

5’-TCGGCTCTCGCATCGATGAAG-3’(SEQ ID No.4)，

5’-GCGCAACTTGCGTTCAAAGAC-3’(SEQ ID No.5)；

the nucleotide sequence of the probe for detecting the reference gene is as follows:

5’-ATGGTTCGCGGGATTCTGCAATTC-3’(SEQ ID No.6)。

because the 5.8S gene has higher conservation, the 5.8S gene is used as an internal reference gene, and the primer and the probe for detecting the internal reference gene are designed. The 5 'end of the probe for detecting the reference gene is also marked with a fluorescent group, and the 3' end of the probe for detecting the reference gene is also marked with a quenching group. For actual detection, the probe for detecting the specific gene and the probe for detecting the reference gene are different in the fluorescent group marked at the 5' end.

A kit for quantitatively detecting dendrobium officinale comprises the detection tool.

A micro-drop digital PCR method for quantitatively detecting dendrobium officinale comprises the following steps:

(1) extracting sample DNA;

(2) adding a primer and a probe in the kit, ddPCR Super Mix and distilled water by taking the sample DNA as a template to prepare a reaction system;

(3) adding the reaction system prepared in the step (2) and the droplet-generating oil into a droplet-generating card, and placing the droplet-generating card in a droplet generator to generate droplets;

(4) and carrying out amplification reaction, and carrying out microdroplet fluorescence data analysis after the reaction is finished.

Preferably, the reaction system of step (2) comprises the following components: 10. mu.L of 2 XDdPCR Super Mix, 1.8. mu.L of each of the upstream and downstream primers, 0.5. mu.L of the 10. mu. mol/L probe, 2. mu.L of the sample DNA, and a solution of sterile distilled water to 20. mu.L.

Preferably, the final concentration of the single primer in step (2) is 600-1200nmol/L, and the final concentration of the single probe is 150-400 nmol/L.

Preferably, the reaction procedure of the amplification reaction in step (4) is: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s, annealing and extension at 60 ℃ for 1min, and amplification for 40 samples; the enzyme was inactivated at 98 ℃ for 10 min.

Compared with the prior art, the invention has the following beneficial effects:

the research adopts a double-channel method, namely different fluorescent labels are carried out on probes for detecting endogenous gene ZW-5.8S and specific gene Dendrobium officinale ITS (TPSH-ITS), the number of molecules of the endogenous gene and the specific gene is simultaneously measured in the same PCR reaction system, and a quantitative detection method based on a micro-drop digital PCR technology is established, is applied to the rapid identification and analysis of Dendrobium officinale and products thereof, has the characteristics of high efficiency, accuracy and rapidness, provides a more powerful basis for the detection and supervision work and the relevant departments to try out and enforce law, and has great significance for improving the quantitative detection capability of Chinese medicinal material components, standardizing the production behaviors of relevant enterprises and ensuring the market safety of the Chinese medicinal materials.

Drawings

FIG. 1 shows the results of the specific assay in example 4;

FIG. 2 is a standard curve fit to the linear range of Dendrobium officinale Kimura et Migo as detected in example 5, wherein FIG. 2A is a standard curve fit to an internal reference gene ZW-5.8S, and FIG. 2B is a standard curve fit to a specific gene TPSH-ITS of Dendrobium officinale Kimura et Migo;

FIG. 3 shows the result of the digital PCR detection of Dendrobium officinale with 90% content in example 7, wherein channel 1 is used for detecting TPSH-ITS and channel 2 is used for detecting ZW-5.8S.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Experimental materials: dendrobium officinale TPSH-01 is collected from Zhejiang, Dendrobium huoshanense HSSH-01 is collected from Anhui province, Dendrobium devonianum CBSH-01 is collected from Guangxi, Dendrobium moniliforme XJSH is collected from Zhejiang, Dendrobium moniliforme DGSH is collected from Yunnan, Dendrobium kojimi QJSH is collected from Sichuan, and Dendrobium candidum MHSH is collected from Guangdong.

Main reagents and instruments: a constant temperature incubator (Eppendorf, germany); high speed refrigerated centrifuge (Eppendorf, germany); KingFisher mL nucleic acid automatic extractor (Saimer Feishel, USA); a NanoDrop1000 micro uv spectrophotometer (seimer feishel, usa); Bio-Rad QX200TM Droplet Digital PCR System (including a Droplet generation card, a Droplet generator, a membrane sealer, a gradient PCR machine, and a Droplet fluorescence detector) (Burley, USA); pipette (Ebende, Germany). CTAB lysate (20g/L CTAB, 1.4mol/L NaCl); magnetic bead adsorption buffer (5g/L CTAB, 40mmol/L NaCl); magnetic bead suspensions (luoyang epson biotechnology limited); TE buffer (10mmol/L Tris-HCl, 1mmol/L disodium EDTA, pH 8.0); 75% alcohol; 2 x ddPCR supermix for probes, droplet generation oil for probes, droplet analysis oil (ddPCR droplet reader oil) (Burley, USA).

Example 1

The embodiment provides a detection tool, which comprises a specific primer and a specific probe for detecting dendrobium officinale, and the inventor uses molecular biology software to design the specific primer and the specific probe by analyzing a specific conserved region of the dendrobium officinale which is different from other similar plant species based on an ITS (internal transcribed spacer) sequence.

Wherein the nucleotide sequence of the designed specific primer is as follows:

5’-ACACTTCTTGGAAACGCCGTC-3’(SEQ ID NO.1)，

5’-CGTACAGAATACGATATCCCTC-3’(SEQ ID NO.2)；

wherein the nucleotide sequence of the designed specific probe is as follows:

5’-CCTCCCTATCGTGGGACAGACAG-3’(SEQ ID NO.3)。

the length of the fragment amplified by using the specific primer is 98 bp.

The 5 'end of the specific probe is marked with a fluorescent group FAM, and the 3' end of the specific probe is marked with a quenching group BHQ 1.

Example 2

The embodiment provides a kit for quantitatively detecting dendrobium officinale, which comprises the detection tool in the embodiment 1, and also comprises a primer and a probe for detecting an internal reference gene (ZW-5.8S gene);

wherein the nucleotide sequence of the primer for detecting the reference gene is as follows:

5’-TCGGCTCTCGCATCGATGAAG-3’(SEQ ID No.4)，

5’-GCGCAACTTGCGTTCAAAGAC-3’(SEQ ID No.5)；

wherein the nucleotide sequence of the probe for detecting the reference gene is as follows:

5’-ATGGTTCGCGGGATTCTGCAATTC-3’(SEQ ID No.6)。

the length of the fragment obtained by amplification using the primer for detecting the reference gene is 96 bp.

The probe for detecting the internal reference gene is characterized in that a fluorescent group HEX is marked at the 5 'end, and a quenching group BHQ1 is marked at the 3' end. Because the 5.8S gene has higher conservation, the 5.8S gene is used as an internal reference gene, and the primer and the probe for detecting the internal reference gene are designed.

Example 3

The embodiment provides a micro-drop digital PCR method for quantitatively detecting dendrobium officinale, which comprises the following steps:

(1) extracting sample DNA;

(2) taking sample DNA as a template, and adding the primer and the probe of the kit in the embodiment 2, ddPCR Super Mix and distilled water to obtain a reaction system;

(4) and carrying out amplification reaction, carrying out microdroplet fluorescence data analysis after the reaction is finished, and calculating to obtain the nucleic acid concentration of the sample.

The method for extracting the sample DNA is not limited, and a CTAB method, a glass bead method, an ultrasonic method, a grinding method, a freeze-thaw method, a guanidinium isothiocyanate method or an alkaline lysis method can be selected.

Wherein the reaction system of the step (2) comprises the following components: 2 XDddPCR Super Mix 10. mu.L, 1.8. mu.L each for upstream and downstream primers, 10. mu. mol (final concentration in reaction system 900nmol/L), 0.5. mu.L each for probe, 10. mu. mol/L (final concentration in reaction system 250nmol/L), 2. mu.L of sample DNA, and make up to 20. mu.L with sterilized distilled water.

The specific operation of the step (3) is as follows: the 20 u L reaction system and 70 u L mineral oil are added into the corresponding hole of the microdroplet generation card together, microdroplets are automatically generated on a microdroplet generator, then the generated 40 u L water-in-oil microdroplets are carefully transferred to a 96-hole reaction plate, the 96-hole reaction plate is sealed on a membrane sealing instrument, and finally, the amplification reaction is carried out on a gradient PCR instrument.

The reaction procedure of the amplification reaction in the step (4) is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s, annealing and extension at 60 ℃ for 1min, and amplification for 40 samples; the enzyme was inactivated by holding at 98 ℃ for 10 min. After the amplification is finished, fluorescence analysis is carried out in a micro-drop digital PCR fluorescence analysis system, and the following settings are carried out in software connected with an instrument: and selecting an absolute quantitative method, setting the specific gene channel as a FAM fluorescence channel, setting the internal reference gene channel as a HEX fluorescence channel, and performing fluorescence data analysis.

Example 4

Specificity detection

Samples specifically verified by the real-time fluorescent PCR method (known as a specific species of sample) were selected, and ddPCR (droplet digital PCR) specific amplification was performed according to the detection method in example 3, thereby verifying the specificity of the detection method.

The detection results are shown in fig. 1, the specific primers and the specific probes in example 1 can effectively amplify dendrobium officinale sample TPSH-01 (corresponding to a02), and the samples to be detected, namely HSSH-01, CBSH-01, XJSH, DGSH, QJSH and MHSH (the samples correspond to a05, B05, C05, D05, E05 and F05 respectively) and the control group and the blank control group CK (corresponding to G05) have no signal amplification, which indicates that the method can specifically amplify dendrobium officinale.

Example 5

Sensitive detection

In order to determine the sensitivity of the digital PCR detection system, 5 ng/. mu.L of positive sample DNA was diluted to 5 concentration gradients of 1 ng/. mu.L, 200 pg/. mu.L, 40 pg/. mu.L, 8 pg/. mu.L and 1.6 pg/. mu.L, respectively, and the sensitivity detection was performed by the method of example 3 (the template amount concentration in the reaction system was one tenth of the sample DNA because the sample DNA occupied one tenth of the volume of the reaction system), and 3 replicates were performed for each concentration gradient. The copy number of each reaction is counted, and the sensitivity of the method system is tested. The sensitivity measurements are shown in table 1, and the data in the linear range are fitted to a standard curve as shown in fig. 2.

TABLE 1 Linear Range of the micro-drop digital PCR method for detection of Dendrobium officinale

The data fitting standard curve of fig. 2 is plotted from the actual copy number detection results in table 1. As can be seen from A in FIG. 2, the copy number of the internal reference gene ZW-5.8S is in the interval of 1.53-524.13, and R is a good linearity²0.9999, and the RSD values of all concentration groups are between 1.39% and 16.41%, and are less than 25%; as can be seen from B in FIG. 2, the ITS gene copy number of Dendrobium officinale can show good linearity within the interval of 1.60-521.07, R²0.9999, and RSD values between 1.43% and 15.02%, all less than 25%, were found for all concentration groups. The method is proved to have good quantitative detection capability when the template amount concentration is 0.8-500 pg/mu L.

Example 6

Limit of quantitative detection

And (3) taking the lowest DNA concentration group which can be stably amplified in the linear range verification to carry out the verification Of the Limit Of quantitative detection (LOQ), wherein the Limit Of quantitative detection in the research Of the method is the lowest copy number which can be stably and quantitatively detected in the linear range Of the method. Therefore, LOQ value verification is performed by taking the lowest template amount (i.e., 0.8 pg/. mu.L) of the linear range verification result. Sample DNA with a template amount of 0.8 pg/. mu.L was subjected to 8 parallel digital PCR amplifications, the copy number of the reference gene and the specific gene for each parallel was calculated, the mean value and RSD value were calculated, and the test results are shown in Table 2.

TABLE 2 LOQ verification result of micro-drop digital PCR method for detecting Dendrobium officinale

As can be seen from table 2, the average copy number of 8 parallel amplifications of the internal reference gene ZW-5.8S was 1.78/. mu.l, the RSD was 21.66%, which was less than the defined 25%, and the method satisfied the method requirements, indicating that the minimum copy number of the method for stable quantitative detection of the internal reference gene in the unit system was 35.6 (the minimum copy number was 20 × 1.78 — 35.6 because the reaction system was 20 μ L). The average value of 8 parallel amplification copy numbers of the ITS gene of the dendrobium officinale is 1.53/. mu.L, the RSD is 18.46 percent and is lower than the limited 25 percent, the requirement of the method is met, and the minimum copy number of the DCXC-ITS gene in a unit system which can be stably and quantitatively detected by the method is 30.6 (the minimum copy number is 20 mu.L, so the minimum copy number is 20 multiplied by 1.53 to 30.6).

Example 7

Accuracy verification

Completely drying and mixing the dendrobium officinale and the dendrobium devonianum, respectively obtaining samples with the dendrobium officinale content of 90%, 50%, 10% and 1%, respectively carrying out nucleic acid extraction, diluting the nucleic acid samples to 1 ng/mu L, carrying out micro-drop digital PCR accuracy verification, repeating each group for 8 times, calculating the copy number and the percentage of the specific gene and the internal reference gene, calculating the average value of the percentages, obtaining the actual detection content, calculating the RSD, and verifying the accuracy of the method. The accuracy verification results of the dendrobium officinale digital PCR quantitative detection method are shown in Table 3.

TABLE 3 verification of accuracy of the microdroplet digital PCR method for detection of Dendrobium officinale

As can be seen from table 3, the content of dendrobium officinale is 87.90%, 50.11%, 9.25% and 0.95% when the theoretical content of dendrobium officinale is 90%, 50%, 10% and 1%, the RSD value of the corresponding detection is 3.34%, 4.77%, 8.57% and 17.69%, the relative standard deviation of the three groups of detection results is less than 25%, and the requirement of the test method is met, and fig. 3 shows the accuracy detection result of digital PCR of dendrobium officinale with 90% content. From the above, the method can be used for accurately and absolutely quantitatively detecting the dendrobium officinale sample.

SEQUENCE LISTING

<110> North Arch customs technology center

<120> micro-drop type digital PCR primer, probe, kit and method for quantitatively detecting dendrobium officinale

<130> 1

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence

<400> 1

acacttcttg gaaacgccgt c 21

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence

<400> 2

cgtacagaat acgatatccc tc 22

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence

<400> 3

cctccctatc gtgggacaga cag 23

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<400> 4

tcggctctcg catcgatgaa g 21

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<400> 5

gcgcaacttg cgttcaaaga c 21

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence

<400> 6

atggttcgcg ggattctgca attc 24

Claims

1. A micro-drop digital PCR primer for quantitatively detecting dendrobium officinale is characterized by comprising a specific primer for amplifying ITS gene of the dendrobium officinale, wherein the nucleotide sequence of the specific primer is as follows:

5’-ACACTTCTTGGAAACGCCGTC-3’(SEQ ID NO.1)，

5’-CGTACAGAATACGATATCCCTC-3’(SEQ ID NO.2)。

2. the micro-drop digital PCR probe for quantitatively detecting the dendrobium officinale is characterized by comprising a specific probe targeting an ITS gene of the dendrobium officinale, wherein the nucleotide sequence of the specific probe is as follows:

5’-CCTCCCTATCGTGGGACAGACAG-3’(SEQ ID NO.3)。

3. the digital PCR probe of claim 2, wherein the specific probe is labeled with a fluorophore and a quencher, and the fluorophore is preferably one of FAM, TET, HEX, CY3, JOE, VIC, ROX, or CY 5; the quenching group is preferably one of BHQ1, BHQ2, TAMRA, DABCYL, MGB or Eclipse.

4. A detection kit comprising the primer of claim 1 and/or the probe of any one of claims 2 to 3.

5. The detection tool according to claim 4, further comprising a detection material for an internal reference gene, wherein the internal reference gene is ZW-5.8S gene.

6. The detection tool according to claim 5, wherein the detection material of the reference gene comprises a primer and a probe for detecting the reference gene;

5’-TCGGCTCTCGCATCGATGAAG-3’(SEQ ID No.4)，

5’-GCGCAACTTGCGTTCAAAGAC-3’(SEQ ID No.5)；

5’-ATGGTTCGCGGGATTCTGCAATTC-3’(SEQ ID No.6)。

7. a kit for quantitatively detecting Dendrobium officinale, which is characterized by comprising the detection tool of any one of claims 4-6.

8. A micro-drop digital PCR method for quantitatively detecting dendrobium officinale is characterized by comprising the following steps:

(1) extracting sample DNA;

(2) adding the primer and the probe in the kit of claim 7, ddPCR Super Mix and distilled water by taking the sample DNA as a template to prepare a reaction system;

9. The digital PCR method of claim 8, wherein the reaction system of step (2) comprises the following components: 10. mu.L of 2 XDdPCR Super Mix, 1.8. mu.L of each of the upstream and downstream primers, 0.5. mu.L of the 10. mu. mol/L probe, 2. mu.L of the sample DNA, and a solution of sterile distilled water to 20. mu.L.

10. The digital PCR method according to claim 8, wherein the reaction procedure of the amplification reaction in step (4) is: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s, annealing and extension at 60 ℃ for 1min, and amplification for 40 samples; the enzyme was inactivated at 98 ℃ for 10 min.