CN113930524B

CN113930524B - Primer, kit and detection method for rapid detection of Chinese medicinal material sparrow brain components

Info

Publication number: CN113930524B
Application number: CN202111309124.4A
Authority: CN
Inventors: 朴晋华; 董培智; 杨宝; 李忠华; 连云岚; 马宁
Original assignee: Shanxi Inspection And Testing Center Shanxi Institute Of Standard Measurement Technology
Current assignee: Shanxi Inspection And Testing Center Shanxi Institute Of Standard Measurement Technology
Priority date: 2021-11-06
Filing date: 2021-11-06
Publication date: 2023-11-28
Anticipated expiration: 2041-11-06
Also published as: CN113930524A

Abstract

The invention relates to a primer, a kit and a detection method for rapidly detecting components of Chinese medicinal materials sparrow brains. Visual observation of the nucleic acid detection result can be realized through the nucleic acid thin film chromatography detection test strip. The invention detects the components of the Chinese medicinal material sparrow brain through the nucleic acid detection test paper, and a single sample can obtain a detection result only after 65-70min from pretreatment to detection, and has no non-specific reaction with other common animal-derived components.

Description

Primer, kit and detection method for rapid detection of Chinese medicinal material sparrow brain components

Technical Field

The invention relates to a primer, a kit and a detection method for rapid detection of components of Chinese medicinal materials, belonging to the technical field of biological detection.

Background

The sparrow brain is prepared by processing fresh brain tissue of sparrow (Passer montanus saturatus Stejneger) of the family Underwort, and is recorded in Ming's medical miscellaneous records, ben Cao gang mu (compendium of materia Medica), ben Cao (diet therapy materia Medica), ben Cao (animal materia Medica), yunnan Ben Cao (Yunnan Ben Cao), modern health care Chinese medicinal dictionary, chinese animal emotion, chinese big dictionary, health care of meat, egg and milk, chinese Ben Cao (Chinese materia Medica) and the like. The sparrow brain has sweet taste, flat nature, kidney meridian tropism, kidney tonifying, yang invigorating, skin moisturizing and granulation promoting effects, and can be used for treating kidney deficiency, impotence, deafness, purgation, chilblain, etc., and can be applied to affected parts for treating rhagadia manus and foot, chilblain, etc.; it should be taken orally for kidney deficiency. The application preparation is a tortoise plastron capsule and a right return pill.

The Queenshan is a cold-prepared medicinal material, the detection method has little research, and the Queenshan content is specified as total nitrogen in the Chinese medicinal material standard of Shanxi province, and the Queenshan is rich in protein and amino acid. The current literature reports that the quality control of the sparrow brain comprises overall quality control and quality control of effective components, wherein the overall quality control comprises the items of character identification, microscopic identification, thin layer identification, moisture and ash inspection, total nitrogen, amino acid and the like. The character identification characteristic of the sparrow brain is that the color and the smell are basically the same as those of the meat meal; microscopic identification is characterized by dendritic fragments and nerve fibers, but is a general feature of any non-lower biological brain tissue; the content measurement and the thin-layer detection of amino acids and the identification of alanine components are not specific, the amino acids are the most basic components of organisms, the alanine is a ubiquitous amino acid, and biological tissues including fermentation liquor and hair hydrolysate have amino acids and alanine; moisture and ash are general examination items of medicinal materials; total nitrogen is widely present in nature, especially in organisms, and even in air. Active ingredient quality control alanine, a ubiquitous amino acid in organisms as previously described, was determined by pre-column derivatization HPLC. Therefore, the quality of the sparrow brain cannot be truly identified or controlled by the whole quality control index or the current single effective component control index, and the brain tissues of partial organisms and other non-lower organisms added into the sparrow brain meet the above standards, so that the current identification and detection method of the sparrow brain is not strong in specificity. According to literature calculations, amino acid analysis can detect a minimum of 2.0. Mu.g of sample, HPLC derivatization can detect a minimum of 0.5. Mu.g of sample, and thin layer chromatography can detect a minimum of 80. Mu.g of sample.

With the intensive research and rapid development of molecular biology in recent years, the method for actually verifying the authenticity of traditional Chinese medicinal materials has been transferred to the molecular biology method such as the common PCR technology, the real-time fluorescence PCR technology, the PCR-RFLP technology and the like through the traditional single physicochemical experiment level. The polymerase chain reaction selectively carries out in vitro simulated cell internal amplification DNA or RNA fragment method through specific primers, the DNA replication method can realize exponential amplification of a very small amount of specific gene fragments in genome in a few hours, the detection sensitivity is remarkably improved, the method becomes the most sensitive detection technology on the current research level, the method has the advantages of high accuracy and strong specificity, and the method plays an important role in various fields such as traditional Chinese medicine authenticity detection and gene mutation detection. However, no primer suitable for PCR detection of the components of the Chinese medicinal material, namely the sparrow brain, is found at present, and the common PCR detection technology has limitations, electrophoresis detection is needed to observe the detection result, and the price of the real-time fluorescence PCR detection equipment is 5 to 10 times that of the common PCR detection equipment, so that the factors limit the application of the real-time fluorescence PCR detection equipment in field detection and basic areas with lack of facilities.

Therefore, the establishment of a rapid, sensitive and specific detection method is essential for guaranteeing the medication safety of people. There is a need to establish a rapid, effective and exclusive method for detecting components of the sparrow brain, which has positive significance in normalizing the harvest processing, market circulation, clinical medication of the medicinal material and quality control of related corresponding preparations.

Disclosure of Invention

First, the technical problem to be solved

In order to solve the problems in the prior art, the invention provides a primer, a kit and a detection method for rapidly, accurately and sensitively detecting the nucleic acid of the component of the sparrow brain.

(II) technical scheme

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:

the primer for detecting the components of the Chinese medicinal material sparrow brain comprises sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.

The rapid detection kit for the components of the Chinese medicinal material sparrow brain comprises the primer pair.

The rapid detection kit for the components of the traditional Chinese medicine sparrow brain comprises the primer pair and a nucleic acid detection test strip, wherein FITC or biotin is marked at the 5 'end of a sequence shown in SEQ ID NO.1, digoxin is marked at the 5' end of a sequence shown in SEQ ID NO.2, the nucleic acid detection test strip is a colloidal gold test strip with a goat anti-FITC antibody/goat anti-biotin antibody coated on a detection line and a goat anti-mouse antibody and a mouse anti-digoxin antibody marked on a colloidal gold coated on a quality control line.

The rapid detection kit for the components of the Chinese medicinal material sparrow brain preferably further comprises PCR Prime STAR HS Premix and detection diluent, wherein the detection diluent comprises 0.3mol/L NaCl and 30mmol/L citric acid buffer solution, and the pH value is 7.0.

The rapid detection kit for the components of the Chinese medicinal material sparrow brain preferably further comprises a DNA extraction reagent, wherein the DNA extraction reagent comprises a lysate, a cleaning solution and a DNA solvent,

the lysate comprises 1.0mol/L guanidine hydrochloride, 1% Triton X-100,1.5% PVP,150mmol/L NaCl,0.5% polysorbate-20, 5mmol/L EDTA,1.5% polyethylene glycol, and PBS solution with pH of 7.4 as solvent, wherein the DNA solvent is TE buffer.

The cleaning solution is formulated as PBS containing 0.15% polysorbate-20 and has a pH of 7.4.

A rapid detection method of components of Chinese medicinal material sparrow brain comprises the following steps:

(1) Extracting sample DNA;

(2) Performing PCR amplification on the DNA template obtained in the step (1) by adopting the primers on the upper and the lower stream of the sequences shown as SEQ ID NO.1 and SEQ ID NO. 2; wherein, the 5 'end of the upstream primer is marked with FITC or biotin, and the 5' end of the reverse primer is marked with digoxin;

(3) And (3) judging the result of the PCR amplification product obtained in the step (4) by using a nucleic acid detection test strip.

As described above, in the rapid test method, preferably, in the step (1), the method of extracting DNA is that the processed sample is ground into powder or the fresh sparrow brain sample is ground into powder or paste 0.02-0.05 g, put into a 2mL centrifuge tube, 500. Mu.L of lysate is added to lyse the sample for 7s, one end of the filter paper strip is immersed in the lysate for 3s to adsorb nucleic acid, and then the filter paper strip is transferred into 1750. Mu.L of washing liquid for 3s to wash impurities adsorbed on the surface; finally, transferring the filter paper strips into prepared 50 mu L TE buffer solution for 1min to elute the nucleic acid adsorbed on the surface; the lysate comprises 1.0mol/L guanidine hydrochloride, 1% Triton X-100,1.5% PVP,150mmol/L NaCl,0.5% polysorbate-20, 5mmol/L EDTA,1.5% polyethylene glycol, and PBS solution with pH of 7.4 as solvent;

the formula of the cleaning solution is PBS with the concentration of 0.15% polysorbate-20 and the pH value is 7.4.

The detection method as described above, preferably, in step (2), the amplification system comprises: PCR Prime STAR HS Premix 25. Mu.L, 10. Mu. Mol/L concentration of each of the upstream and downstream primers 0.25. Mu.L, 1. Mu.L of the template, and water to 50. Mu.L;

the PCR amplification reaction conditions are as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 63 ℃ for 30s,30 cycles, and extending at 72 ℃ for 5min.

In the above detection method, preferably, in the step (3), when the result of the PCR amplification product is determined by electrophoresis, the amplification product is electrophoretically imaged, and if the amplification product has a band of 336bp, it is indicated that the sample contains a sparrow brain component; if no amplified band exists, the sample does not contain the component of the sparrow brain.

In the above detection method, preferably, in the step (3), the nucleic acid detection test strip is a colloidal gold test strip coated with goat anti-FITC antibody/goat anti-biotin antibody, a quality control line is coated with goat anti-mouse antibody and mouse anti-digoxin antibody, and after the amplified product is diluted with a detection diluent, the amplified product is dripped into a sample adding place of the nucleic acid detection test strip for detection, wherein the detection diluent is prepared from a citric acid buffer solution containing 0.3mol/L NaCl and 30mmol/L and has a pH of 7.0.

In the rapid detection method, preferably, the sample is added and then interpreted for 5-8 minutes, so as to obtain a corresponding detection result, and the interpretation at other times is invalid; in the course of the reading of the result,

when the detection line T line and the quality control line C line are both developed, the sample is indicated to contain the sparrow brain component;

when the detection line T line does not develop and the quality control line C line develops, the sample does not contain the sparrow brain component; when the quality control line C does not develop color, the operation is not indicated or the test paper is failed, and the detection should be repeated.

(III) beneficial effects

The beneficial effects of the invention are as follows:

according to the method, after the specific primers of the sparrow brains are added with the markers, DNA of the sparrow brains samples is amplified, the amplified products can be specifically combined with the detection test strips, experiments prove that the amplified products are free of nonspecific combination with the common 16 animal brains, the specificity is very strong, 0.008 mug of samples can be detected at the lowest, the actual sensitivity is higher than that of the conventional detection technologies, the detection time is only 65-70 minutes, the test can be completed completely, the result is obtained, the efficiency is greatly higher than that of the conventional integral quality control method, at least 3 days are required, and the liquid phase method requires 5 hours of aging.

According to the PCR amplification technology and the more efficient molecular detection method for detecting the components of the sparrow brain by combining the nucleic acid detection test strip, the optimal amplified fragment position and length can be effectively avoided from being formed by non-specific amplification through the design selection of the primer, and the optimal development buffer solution is optimized to enable the nucleic acid test strip to achieve the optimal color development effect. The detection method does not pollute laboratories and surrounding environments, does not need expensive instruments and equipment or professional operating skills, and can be used for detecting large-scale samples under the condition of imperfect experimental conditions, and the specific advantages are as follows:

1. the nucleic acid detection test strip for detecting the amplification product of the sparrow brain component has the advantages of strong specificity, high sensitivity, simple operation and strong stability;

2. the amplification process is carried out in a closed reaction system, so that the pollution of amplification products is effectively avoided;

3. the reaction speed is high, and the whole detection steps of the sample are completed only for about 1 hour;

4. the detection process does not need complicated and expensive instruments and equipment and is operated by professional technicians;

5. is suitable for the preliminary judgment of the on-site rapid detection of the components of the Chinese medicinal material sparrow brains.

The establishment of the nucleic acid detection test strip can realize the visual observation of the nucleic acid detection result. The invention detects the components of the Chinese medicinal material sparrow brain through the nucleic acid detection test paper, and a single sample can obtain a detection result only after 1 hour from pretreatment to detection, and has no non-specific reaction with other common animal-derived components.

Drawings

FIG. 1 shows the result of electrophoresis detection of a general PCR for primer selection;

FIG. 2 shows the detection results of fluorescence quantitative PCR for primer selection;

FIG. 3 shows the amplification results of annealing temperature selection of common PCR primers;

FIG. 4 shows the results of the specificity verification of the common PCR on the brain components of 16 animals;

FIG. 5 shows the results of specific verification of fluorescent quantitative PCR on brain components of 16 animals;

FIG. 6 is a schematic diagram showing a test strip for nucleic acid detection;

FIG. 7 shows the results of the dilution of the sparrow brain component;

FIG. 8 shows the result of specific detection by the nucleic acid detection test strip method;

FIG. 9 shows the detection of the components of a commercial sample of the sparrow brain by using a nucleic acid test strip;

FIG. 10 shows the result of the sensitivity of the conventional PCR detection;

FIG. 11 shows the results of the sensitivity of the self-made nucleic acid test strip.

Detailed Description

The invention principle of the invention: in the PCR amplification process, two different types of nucleic acid markers which can be captured by a specific binding antibody or a ligand thereof are introduced into an amplification product by utilizing an upstream primer and a downstream primer respectively modified with a specific marker, and the detection principle is that FITC/biotin-target fragment-digoxin generated in the amplification step is combined with a mouse anti-digoxin antibody coated with colloidal gold particles on test paper to form a FITC/biotin-target fragment-digoxin-mouse anti-digoxin antibody-chromogenic particle complex according to a colloidal gold immunochromatography analysis method of a double antibody sandwich method; the obtained chromogenic particle compound moves upwards to a detection line along an NC film by capillary action in a loading buffer solution and is captured by a receptor goat anti-FITC antibody/goat anti-biotin antibody to obtain a goat anti-FITC antibody/goat anti-biotin antibody-FITC/biotin-target fragment-digoxin-mouse anti-digoxin antibody-chromogenic particle compound, a macroscopic chromogenic strip is formed by sedimentation on the detection line, and a gold-labeled antibody coated on colloidal gold particles which are not combined with an amplification product is combined with a goat anti-mouse antibody on a quality control line, wherein the two chromogenic strips are positive results; when the nucleic acid amplification product of the sparrow brain component does not exist, the steps do not occur, the sparrow brain component cannot be combined with the digoxin antibody coated with the colloidal gold particles on the gold-labeled pad (glass fiber), the chromogenic substance colloidal gold particles cannot be combined with the substance coated on the detection line to form a chromogenic strip, and the goat anti-mouse antibody on the quality control line is combined with the gold-labeled antibody coated on the colloidal gold particles to form a chromogenic strip, namely a negative result.

The newly developed DNA extraction reagent can extract a DNA template rapidly and effectively, and the action of salt in the lysate not only provides a proper cracking environment, but also ensures that Triton X-100 is used for separating phospholipids in brain tissues so that the DNA is easier to release, EDTA can inhibit the nuclease in a sample from damaging nucleic acid in the cracking process, and NaCl is used for maintaining the stability of the nucleic acid structure; guanidine hydrochloride can be used for cleaning impurities adsorbed on the surface by denaturing proteins, destroying membrane structures and untangling proteins connected with nucleic acids, so that the nucleic acids are dissociated in a lysis system. The DNA extraction reagent provided by the invention can be used for rapidly and effectively extracting the sparrow brain DNA template, and the purpose of extracting target DNA can be realized only by 30 seconds.

The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.

Example 1

(1) Primer design

Upstream primers were used in the early experiments: LCO1490 (SEQ ID NO. 3) 5'-GGT CAACAAATCATAAAGATATTGG-3', downstream primer: HCO2198 (SEQ ID No. 4) 5'-TAAACTTCAGGGTGACCAAAAAATCA-3', (synthesized by Shanghai biosciences limited) was denatured at 94 ℃ for 5min;94 ℃,30 s,56 ℃,30 s,72 ℃ and 1min, and reacting for 40 cycles; extending at 72℃for 10min. Sequencing the amplified products obtained from the samples (7 batches of samples collected as described in example 5) and searching NCBI database to obtain sequences (CO 1 sequences) of cytochrome oxidase 1 genes of sparrow, quail, chicken, partridge, pigeon, goose, duck, parrot, turkey and other common proximal species, and comparing the sequences searched by the 7 sequencing samples and NCBI to ensure that the sequences have no mutation sites at the primer, the obtained target gene sequence is shown as SEQ ID NO.5,

the Primer sequence is firstly designed by using Oligov7.0.1 and NCBI Primer-Blast, the obtained Primer sequence is compared by Snapgene software, and the Primer sequence with the largest difference between species is designed to be used for identifying the authenticity of the sparrow brain, and the PCR detection Primer comprises the following primers of common PCR detection and real-time fluorescence PCR detection.

SEQ ID NO.5：

TAAACTTCAGGGTGACCAAAAAATCAACCCTGTACCTAATCTTCGGCGCATGAGCCGGGATAGTAGGTACCGCTCTAAGCCTACTTATCCGAGCAGAACTTGGACAACCAGGGGCTCTCCTAGGAGATGACCAAGTTTACAACGTAGTAGTTACAGCTCACGCTTTCGTGATAATCTTCTTCATAGTTATGCCAATTATGATCGGAGGATTCGGAAACTGACTAGTCCCCCTAATAATTGGAGCACCAGATATAGCATTCCCACGAATAAACAACATAAGCTTCTGACTACTGCCTCCATCCTTCCTCCTGCTACTAGCATCCTCCACCGTAGAAGCAGGAGCAGGCACAGGATGAACAGTATACCCCCCACTGGCCGGCAACCTAGCCCACGCCGGAGCCTCAGTAGATCTAGCAATCTTCTCCCTACACTTAGCAGGTATCTCATCAATCTTAGGGGCAATCAACTTTATTACAACAGCAATCAACATAAAACCCCCCGCCCTATCACAATATCAAACACCCCTATTTGTCTGATCCGTACTAATCACCGCAGTACTACTGCTCCTGTCGCTACCCGTCCTCGCTGCAGGAATCACAATGCTACTCACCGACCGCAACCTCAACACCACATTCTTTGACCCCGCAGGCGGAGGAGATCCAGTCCTATACCAACATCTCTTCCCAATATCTTTATGATTTGTTGACC。

Common PCR detection:

sequences of group 1 primers

Forward primer (SEQ ID No. 6): 5'-ACAGCTCACGCTTTCGTGATA-3'

Reverse primer (SEQ ID NO. 7): 5'-GGGTAGCGACAGGAGCAGTA-3'

Sequences of group 2 primers

Forward primer (SEQ ID No. 1): 5'-GCTTCTGACTACTGCCTCCATC-3'

Reverse primer (SEQ ID NO. 2): 5'-GGTCGGTGAGTAGCATTGTG-3'

Real-time fluorescent PCR detection

Sequences of group 3 primers

Forward primer (SEQ ID No. 8): 5'-CACCAGATATAGCATTCCCACGAA-3'

Reverse primer (SEQ ID NO. 9): 5'-ATACTGTTCATCCTGTGCCTGC-3'

Sequences of group 4 primers

Forward primer (SEQ ID No. 10): 5-AGCACCAGATATAGCATTCCCAC-3'

Reverse primer (SEQ ID NO. 11): 5'-CTGCTCCTGCTTCTACGGTG-3'

Real-time fluorescent PCR probe (SEQ ID NO. 12): FAM5'-TCTGACTACTGCCTCCATCCTTCC-3' TAMRA

(2) And (3) performing PCR reaction detection: sparrow brain PCR amplification reaction system: PCR Prime STAR HS Premix (available from Takara Bio Inc.) 25. Mu.L, 10. Mu. Mol/L concentration of each of the upstream and downstream primers 0.25. Mu.L, 1. Mu.L of the template, and water to 50. Mu.L.

Real-time fluorescent PCR amplification system: ABI TaqMan Fast Universal PCR Master Mix (2X) 12.5. Mu.L each of the upstream and downstream primers at a concentration of 10. Mu. Mol/L, the probe 0.25. Mu.L, the template 1. Mu.L, and the water was supplied to 25. Mu.L.

Wherein, the PCR amplification reaction conditions are as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 63 ℃ for 30s,30 cycles, and extending at 72 ℃ for 5min.

The real-time fluorescent amplification conditions are: pre-denaturation at 95℃for 2min; denaturation at 95℃for 15s, annealing at 60℃for 30, 40 cycles.

A1.5% agarose Gel was prepared with a 1 XTAE solution and Gel red was added to a final concentration of 0.5. Mu.g/mL. An electrophoresis buffer is added into the electrophoresis tank, so that the liquid surface just gets out of the gel. 3 μL PCR amplification product was added to each well and 1.5 μL DNA Marker was added to the first spotted well. After electrophoresis until bromophenol blue indicator migrates to gel 2/3 at constant pressure of 5V/cm, the result of electrophoresis was observed under a gel imager and recorded.

The result of the common PCR electrophoresis is shown in FIG. 1, wherein the 1-bit primer amplification result is shown in FIG. 1, 2 is the second group amplification result, K is blank control, and M is DNAmark. In the gel electrophoresis pattern, the amplification products of the 1 st group of primers have obvious amplification product bands between 400 and 500bp, the actual reference fragment length is 426bp, and the experimental result is consistent with the actual reference fragment, but has weak non-specific amplification bands; the amplification product of the 2 nd group of primers has obvious amplification product bands between 300 and 400bp, the actual reference fragment length is 336bp, the experimental result is consistent with the actual reference fragment length, and the primer 2 has no non-specific amplification band, so the 2 nd group of primers is selected as the optimal primer for PCR amplification.

The real-time fluorescence PCR result is shown in figure 2, the blue amplification curve is the 4 th group primer sequence, the red amplification curve is the 3 rd group primer sequence, the same Taqman probe is adopted for the amplification of the two primer sequences, the two groups of primers have fluorescence signal detection, a typical amplification curve appears, ct values are less than 35, the amplification efficiency of the 4 th group primer sequence is higher than that of the 3 rd group primer sequence, and therefore the 4 th primer sequence is selected as the optimal primer of the real-time fluorescence PCR method.

Wherein, the amplified products of the group 2 primers are purified and then sequenced, and the sequencing result is shown as the amplified sequence shown as SEQ ID NO. 13: GCTTCTGACTACTGCCTCCATCCTTCCTCCTGCTACTAGCATCCTCCACCGTAGAAGCAGGAGCAGGCACAGGATGAACAGTATACCCCCCACTGGCCGGCAACCTAGCCCACGCCGGAGCCTCAGTAGATCTAGCAATCTTCTCCCTACACTTAGCAGGTATCTCATCAATCTTAGGGGCAATCAACTTTATTACAACAGCAATCAACATAAAACCCCCCGCCCTATCACAATATCAAACACCCCTATTTGTCTGATCCGTACTAATCACCGCAGTACTACTGCTCCTGTCGCTACCCGTCCTCGCTGCAGGAATCACAATGCTACTCACCGACC, the result is accurate.

After purifying the amplified products of the 4 th group of primers, carrying out clone sequencing, wherein the sequencing result is shown as an amplified sequence shown as SEQ ID NO. 14: AGCACCAGATATAGCATTCCCACGAATAAACAACATAAGCTTCTGACTACTGCCTCCATCCTTCCTCCTGCTACTAGCATCCTCCACCGTAGAAGCAGGAGCAG.

(3) Annealing temperature selection of common PCR primer

The optimal annealing temperature is verified by PCR amplification with the primer of the group 2, and the conditions for PCR amplification reaction of the components of the sparrow brain are respectively selected from the annealing temperature of 68 ℃, 67.1 ℃, 65.5 ℃, 63.1 ℃, 60.1 ℃, 58 ℃, 56.2 ℃ and 55 ℃ as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30s; annealing at 55-68 ℃ for 30s, extending at 72 ℃ for 30s,35 cycles, and extending at 72 ℃ for 5min.

The results of the amplified bands after electrophoresis of the amplified products at different annealing temperatures are shown in FIG. 3, where M: DNA molecular weight marker DL1000: the corresponding annealing temperature 1:68 ℃ 2:67.1 ℃ 3:65.5 ℃ 4:63.1 ℃ 5:60.1 ℃ 6:58 ℃ 7:56.2 ℃ 8:55 ℃. Depending on the amplified band quality, 63℃was finally selected as the annealing temperature.

Example 2 verification of specificity of amplified products to brain tissue of different animals

(1) Sample pretreatment extraction

Taking 16 animal brain samples (16 animals brain tissue 1: sparrow 2: pigeon 3: goose 4, pig 5: donkey 6: fox 7: salmon 8: black tail snake 9: quail 10: chicken 11: rabbit 12: sheep 13: duck 14: cow 15: horse 16: marten K) 20mg, respectively grinding into powder or paste for extracting DNA. The extraction can be performed by using a DNA extraction kit, and the following method can also be adopted: (2) a common PCR amplification reaction system adopts: PCR Prime STAR HS Premix (available from Takara Bio Inc.) 25. Mu.L, and the sequence of the upstream and downstream primers of group 2 in example 1 were set to 0.25. Mu.L each of SEQ ID NO.1 and 2 (concentration 10. Mu. Mol/L), and template 1. Mu.L was made up to 50. Mu.L. The PCR amplification reaction conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 63 ℃ for 30s,30 cycles, and extending at 72 ℃ for 5min.

(3) The specificity of the amplified product is verified by using a common PCR electrophoresis method to form a knotThe result is shown in FIG. 4, where M: DNA molecular weight marker DL1000; brain tissue 1 of the corresponding 16 animals: sparrow 2: pigeon 3: goose 4, pig 5: donkey 6: fox 7: salmon 8: zaocys 9: quail 10: chicken 11: rabbit 12: sheep 13: duck 14: cattle 15: horse 16: mink K: blank control (ddH) ₂ O). The results show that: the primer only has specific binding to the components of the sparrow brain, the results of the components of the sparrow brain are positive, and the results of the DNA components of other brain tissues are negative. Indicating that the primers used for amplification are highly specific.

The above samples were subjected to real-time fluorescent PCR amplification as shown in FIG. 5, and the results were 1, sparrow (sparrow), 2, pigeon (pipe), 3, goose (goose), 4, pig (porcine), 5, donkey (donkey), 6, fox (fox), 7, salmon (salcon), 8, zaocys dhumnades (zaocoys), 9, quail (corernix), 10, chicken (chicken), 11, rabbit (rabbit), 12, sheep (ovine), 13, duck (duck), 14, cow (bovine), 15, horse (horse), 16, mink (marten) K). Blank control (ddH) ₂ O). The results show that: the primer does not generate nonspecific amplification in 35 cycles, the real-time fluorescent PCR is easy to generate cross reaction after 35 cycles, and when CT is more than 35, nonspecific amplification influence result judgment can be generated, so that the detection result of the common PCR is more accurate, the cost of reagents or instrument and equipment is far lower than that of the real-time fluorescent PCR, and the detection technology is more convenient to popularize.

Example 3

In order to extract DNA rapidly from the sample, reagents for rapid DNA extraction were developed, which included a lysate and a washing solution, wherein the lysate was formulated to contain 1.0mol/L guanidine hydrochloride, 1% Triton X-100,1.5% PVP,150mmol/L NaCl,0.5% polysorbate-20, 5mmol/L EDTA,1.5% polyethylene glycol, and the solvent was PBS (pH 7.4). The wash solution formulation was PBS (pH 7.4) containing 0.15% polysorbate-20.

The DNA is adsorbed by a filter paper, the filter paper can be a rectangular filter paper with the diameter of 44mm multiplied by 2mm, or a filter paper with one end immersed with paraffin, and the filter paper is as follows: cutting filter paper into rectangular filter paper strips with the diameter of 44mm multiplied by 2mm by scissors, immersing one end of the filter paper strips into paraffin melted by heating, and forming a hydrophobic handle area with the diameter of 40mm multiplied by 2mm after the filter paper strips are dried in the air so as to transfer the filter paper strips, wherein the part of the front end of the filter paper strips, which is not immersed in the paraffin, is a nucleic acid binding area for subsequent nucleic acid adsorption.

Placing a sample to be tested into a 2mL centrifuge tube, adding 500 mu L of lysate to lyse the sample for 7s, immersing one end of the filter paper strip serving as a nucleic acid binding area into the lysate for 3s to adsorb nucleic acid, and transferring the end of the filter paper strip, which adsorbs DNA, into 1750 mu L of cleaning solution for 3s to clean impurities adsorbed on the surface. Finally, the filter paper strip is transferred into the prepared TE buffer solution for 1min, so that the nucleic acid adsorbed on the surface is eluted, and the nucleic acid enters into the TE solution.

This example uses nucleic acid test paper (FITC-digoxin) purchased from Shanghai Youlong Biotechnology Co.

The sequences of the primers used are shown as SEQ ID NO.1 (forward primer) and SEQ ID NO.2 (reverse primer), and are labeled with FITC at the 5 'end of the forward primer and digoxin at the 5' end of the reverse primer.

The detection method comprises the following steps:

(1) Extraction of sample DNA

Weighing the processed sample, grinding the processed sample into powder or grinding the fresh sparrow brain sample into powder or paste 0.05-0.15 g, placing the powder or paste into a 2mL centrifuge tube, adding 500 mu L of lysate to crack the sample for 7s, immersing a filter paper strip into the lysate for 3s to adsorb nucleic acid, and transferring the filter paper strip into 1750 mu L of cleaning solution for 3s to clean impurities adsorbed on the surface. And finally, transferring the filter paper strips into a prepared amplification reaction system for 3s to elute the nucleic acid adsorbed on the surface, and completing the preparation of the amplification system.

(2) Amplification reaction system of sparrow brain: PCR Prime STAR HS Premix (available from Takara Bio Inc.) 25. Mu.L, FITC-labeled upstream and downstream primers (concentration 10. Mu. Mol/L), template 1. Mu.L, and water to 50. Mu.L. The conditions of the PCR amplification reaction of the components of the sparrow brain are as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 63 ℃ for 30s,30 cycles, and extending at 72 ℃ for 5min.

(3) And dripping the PCR amplification product into a sample area of the nucleic acid detection test strip.

(4) And judging after 5-8 minutes to obtain a corresponding detection result, and judging at other times to be invalid.

(5) When the result is read, the test paper is placed horizontally on the front of the observer.

(6) The result judgment criteria are as shown in figure 6,

negative (-). C line appears, T line does not develop color;

positive (+): c line appears, and the color development of the T line is visible to naked eyes;

invalidation: the C-line is not present and may be mishandled or the test paper has failed.

Dilution factor investigation of amplified products: diluting the amplified products of the components of the sparrow brain by 5 times, 10 times, 15 times and 20 times respectively with detection diluent, wherein the detection diluent comprises the following components: 30mmol/L of citric acid buffer pH7.0 and 0.3mol/L of NaCl. And the sample is added into a detection test strip for detection, the experimental result is shown in figure 7, and when the dilution factor is finally determined to be 15 times, the use amount of amplified products can be saved, and the experimental requirement can be met to obtain an accurate detection result.

Example 4 sparrow brain component nucleic acid test strip specificity experiment

Sample pretreatment: respectively taking 16 animal brain samples (16 corresponding animal brain tissues 1: sparrow 2: pigeon 3: goose 4, pig 5: donkey 6: fox 7: salmon 8: black tail snake 9: quail 10: chicken 11: rabbit 12: sheep 13: duck 14: cow 15: horse 16: marten) 20mg, grinding into powder or paste, placing into a 2mL centrifuge tube, adding 500 μl lysate to crack the sample for 7s, immersing a filter paper strip in the lysate for 3s to adsorb nucleic acid, and transferring the filter paper strip into 1750 μl cleaning solution for 3s to clean impurities adsorbed on the surface. And finally, transferring the filter paper strips into a prepared amplification reaction system for 3s to elute the nucleic acid adsorbed on the surface, and completing the preparation of the amplification system.

(2) An amplification reaction system using the primers of example 3, wherein the system comprises: PCR Prime STAR HS Premix (available from Takara Bio Inc.), 25. Mu.L each of the upstream and downstream primers (concentration 10. Mu. Mol/L), 0.25. Mu.L each of the template, 1. Mu.L, and water to 50. Mu.L. The PCR amplification reaction conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 63 ℃ for 30s,30 cycles, and extending at 72 ℃ for 5min.

(3) Diluting 50 mu L of the PCR amplification product by 15 times with a detection diluent, and putting the diluted product into a nucleic acid detection test strip, wherein the detection diluent comprises the following components: 30mmol/L, pH7.0 citric acid buffer, 0.3mol/L NaCl.

(4) And judging for 5-8 minutes to obtain a corresponding detection result, and judging for other times to be invalid.

The results are shown in FIG. 8, which shows from left to right 1, sparrow, 2, pigeon, 3, goose, 4, pig, 5, donkey, 6, fox, 7, salmon, 8, zaocys, 9, quail, 10, chicken, 11, rabbit, 12, sheep, 13, duck, 14, cow, 15, horse, 16, marten, 17, blank control (ddH ₂ O), the results show that the components of the animals except sparrows are negative, and the nucleic acid detection test strip method has high specificity.

Example 5

There are two common sparrow brain processing modes in the market at present, and one mode is four seasons. When the head is killed, taking out the fresh blood of the head for use or killing, taking out the fresh brain marrow for use, and temporarily defining the fresh sparrow brain; the other is to take out brain marrow after capturing in winter, process with sulfur, process sparrow brain sample with sulfur. 3 batches of fresh sparrow brain samples and 4 batches of sulfur-processed sparrow brain samples are respectively collected, and the samples are collected from the Bozhou traditional Chinese medicine market and are provided by Shanxi Guangdong national medicine Co.

The detection method was carried out by the method in example 4.

The results are shown in FIG. 9, wherein QN-1, 2 and 3 respectively represent 3 batches of collected fresh sparrow brains, and PQN-1, 2, 3 and 4 respectively represent 4 batches of sulfur-collected sulphurous sparrow brain samples. The results show that the components of the sparrow brains can be detected by collecting 3 batches of fresh sparrow brains and 4 batches of sulfur-treated sparrow brain samples.

The nucleic acid detection test strips are used for detecting the samples collected in 7 batches, and the components of the sparrow brain can be detected, so that the method has good repeatability.

Example 6

10ng of the DNA template extracted from the sparrow brain is diluted to 10 times of decrease ^-5 The diluted concentrations were 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng, 0.0001ng, respectively, and 5 replicates were made for each gradientThe experiment was performed according to the PCR amplification method of example 2, using water as a blank control, and the absolute sensitivity of the PCR method was measured.

The method and reagents of example 3 were also used to detect the absolute sensitivity of the nucleic acid test strip.

Samples were subjected to sparrow brain component PCR at 10ng dilutions of 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng, 0.0001ng, and gel electrophoresis results are shown in FIG. 10, wherein 1-5 spotted wells 10ng,6-10 spotted wells 1ng,11-15 spotted wells 0.1ng,16-20 spotted wells 0.01ng,21-25 spotted wells 0.001ng,26-30 spotted wells 0.0001ng, and k as blank controls, and the results indicated that amplified DNA templates of 10ng, 1ng, 0.1ng, 0.01ng, and 0.001ng had a specific amplified band at 336bp in the position corresponding to the electropherogram, and that there was no band in the 0.0001ng and blank controls. The method shows that the common PCR method can still detect the electrophoresis band at 0.001 ng/reaction, and the absolute sensitivity of the common PCR method can reach 0.001 ng/reaction.

In contrast, when the nucleic acid test strip purchased in example 3 was used for detection, only 10ng, 1ng and 0.1ng were detected as positive results, indicating that the absolute sensitivity of detection by the commercially available test strip was 0.1 ng/reaction.

Example 7

As can be seen from example 6, the commercial nucleic acid test strip has low sensitivity, and in order to obtain higher sensitivity, the preparation of the nucleic acid test strip is performed as follows:

preparation of 1-nm gold particles

The trisodium citrate method is adopted to prepare the 25nm nano gold particles. 100mL of a 0.01% chloroauric acid solution (HAuCl) ₄ ) Heated to boiling and then 0.5, 1, 1.5, 2, 2.5, 3 and 4mL of 1% trisodium citrate solution were added, respectively. After boiling for about 5min, the color of the solution changed from yellow to wine red. The solution was then cooled and filtered. Finally, the nano gold solution is stored at 4 ℃. As a result, the color of the gold nanoparticle solution is mainly determined by the amount of trisodium citrate used in the preparation process of gold nanoparticle. With the increase of the addition amount of trisodium citrate, the color of the nano gold solution is changed from light purple to red, but the addition amount is thatAt 2.5mL and 4mL, there was no significant change in red. Thus, 2.5mL of trisodium citrate can be used as the optimal added volume. And finally, adding 2.5mL of the colloidal gold solution obtained by the preparation of the trisodium citrate, and carrying out subsequent experiments.

Preparation of 2-nanometer gold antibody conjugate

The prepared colloidal gold is added in an amount of 0.1mol/L K ₂ CO ₃ After adjusting the pH to 6.6-7.0, the purified mouse anti-digoxin antibody (Roche, germany) was diluted to 0.5mg/mL with phosphate buffer, and then slowly added to 3mL of nano-gold solution for labeling at a labeling concentration of 10. Mu.g/mL. After 20min of reaction, 8. Mu.L of 10% BSA blocking solution was added and the mixture was gently stirred for 10min to stabilize the conjugate. After the resulting solution was centrifuged at 9000rpm for 30min, it was resuspended in 1.5mL of coupling dilution buffer (1% BSA and 0.02M phosphate buffer), and the obtained nanogold antibody conjugate solution was uniformly applied to a glass fiber membrane as a conjugate pad and dried at 37℃for 2h.

3 coating of detection and control lines goat anti-biotin antibody and goat anti-mouse IgG antibody (Sigma, usa) were embedded in detection (T) and control (C) lines of Nitrocellulose (NC) membrane with 0.01M phosphate buffer (pH 7.4), respectively, at antibody concentrations of 1.5mg/mL and 2mg/mL. The treated NC film was dried at room temperature for 10min. A Nitrocellulose (NC) film is attached to the backing card.

4 Assembly of nucleic acid detection test strips

The sample pad, the combining pad and the water absorbing pad are sequentially stuck on the bottom lining card, cut into strips with the width of 0.4cm, the cut test strips are packaged in a shell, and then the test strips and a drying agent are packaged in an aluminum foil bag. Wherein, the quality control line on the NC film is arranged near one side of the absorption pad, the detection line is arranged near one side of the sample pad, the test paper is cut into a detection strip of 4mm by a chopper, and then the detection strip and a drying agent are packaged in an aluminum foil bag. The sample pad and the bonding pad are both made of glass fiber films, and the absorption pad is made of absorbent paper.

A large number of experimental researches show that the non-characteristic strips can be effectively avoided by immersing the sample pad in a BSA solution with the mass and volume percentage of 3% and then drying the sample pad at 50 ℃.

5 test method and judgment standard

The primer sequence is marked during synthesis, biotin is marked at the 5 'end of the forward primer SEQ ID NO.1, and digoxin is marked at the 5' end of the reverse primer SEQ ID NO. 2.

The DNA content was diluted to 10ng of 10 ⁰ ～10 ^-5 The sample is subjected to the PCR detection of the components of the sparrow brain,

in the PCR amplification, the primers (SEQ ID NO.1, SEQ ID NO. 2) were used, and the amplification system and conditions in example 2 were used.

Diluting 50 mu L of the PCR amplification product by 15 times with a detection diluent, and putting the diluted product into a nucleic acid detection test strip, wherein the detection diluent comprises the following components: 30mmol/L, pH7.0 citric acid buffer, 0.3mol/L NaCl.

The detection results are shown in fig. 10, in which fig. 1:10ng,2:1ng,3:0.1ng,4:0.01ng,5:0.001ng,6:0.0001ng,7 is blank. As can be seen in the figure, 1-5 is positive and 6 and 7 are negative. The result shows that the absolute sensitivity of the self-made test strip detection method is 0.001ng.

Example 8 test of sensitivity to detection of artificially prepared Mixed samples

Sparrow brains were added to different matrices and mixed samples were prepared as follows: accurately weighing the bird brain powder by using a ten-thousandth precision analytical balance, adding the bird brain powder into the pig brain freeze-dried powder, and respectively preparing mixed samples with the weight content ratio (bird brain component) of 10%, 1%, 0.1% and 0.01%;

for each group of mixed samples, 3 samples are respectively taken according to each content ratio, the matrix samples with the same amount are synchronously and independently taken to extract nucleic acid, and the DNA extraction method provided by the embodiment 3 of the invention is adopted to respectively detect the DNA extraction method by using the test strip prepared by the embodiment 7. The test results are shown in the following table:

test results of mixed test sample

Adding sparrow brainContent of ingredients	Detection result
		10％	Detection of
1％	Detection of
		0.1％	Detection of
0.01％	Not detected

The result shows that the actual sensitivity of the actual sample mixture can reach 0.1%, and the minimum detection of 0.008 mug sample can be obtained through conversion, and the actual detection sensitivity of the sample is higher than that of the prior art.

From the actual situation of sample doping, the doping can be detected by one thousandth, so that the actual detection requirement is met, and the self-made nucleic acid detection test paper provided by the invention has extremely high detection sensitivity, and can meet the actual detection requirement of trace sparrow brain components.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any person skilled in the art may make modifications or alterations to the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Sequence listing

<110> Shanxi province inspection and detection center (Shanxi province standard metering technology institute)

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Claims

1. A rapid detection kit for Chinese medicinal material sparrow brain components is characterized by comprising a primer pair shown as SEQ ID NO.1 and SEQ ID NO.2,

the kit also comprises a nucleic acid detection test strip, wherein FITC or biotin is marked at the 5 'end of the sequence shown in SEQ ID NO.1, digoxin is marked at the 5' end of the sequence shown in SEQ ID NO.2, and the nucleic acid detection test strip is a colloidal gold test strip with a detection line coated with goat anti-FITC antibody/goat anti-biotin antibody, a quality control line coated with goat anti-mouse antibody and a colloidal gold marked with mouse anti-digoxin antibody;

the kit also comprises PCR Prime STARHS Premix and a detection diluent, wherein the formula of the detection diluent is citric acid buffer solution containing 0.3mol/L NaCl and 30mmol/L, and the pH value is 7.0.

2. The rapid detection kit of claim 1, further comprising a DNA extraction reagent comprising a lysate and a wash solution and a DNA solvent; the formula of the lysate comprises 1.0mol/L guanidine hydrochloride, 1% Triton X-100,1.5% PVP,150mmol/L NaCl,0.5% polysorbate-20, 5mmol/L EDTA,1.5% polyethylene glycol, and PBS solution with pH of 7.4 as solvent; the formula of the cleaning solution is PBS containing 0.15% polysorbate-20, the pH is 7.4, and the DNA solvent is TE buffer solution.

3. A rapid detection method of components of Chinese medicinal material sparrow brain is characterized by comprising the following steps:

(1) Extracting sample DNA;

(3) Judging the result of the PCR amplification product obtained in the step (2) by using a nucleic acid detection test strip;

in the step (1), the method for extracting DNA is that the processed sample is ground into powder or fresh sparrow brain sample is ground into powder or paste 0.02-0.05 g, the powder or paste is placed in a 2mL centrifuge tube, 500 mu L of lysate is added to crack the sample for 7s, one end of a filter paper strip is immersed in the lysate for 3s to adsorb nucleic acid, and then the filter paper strip is transferred into 1750 mu L of cleaning solution for 3s to clean impurities adsorbed on the surface; finally, transferring the filter paper strips into prepared 50 mu L TE buffer solution for 1min to elute the nucleic acid adsorbed on the surface; the lysate comprises 1.0mol/L guanidine hydrochloride, 1% Triton X-100,1.5% PVP,150mmol/L NaCl,0.5% polysorbate-20, 5mmol/L EDTA,1.5% polyethylene glycol, and PBS solution with pH of 7.4 as solvent;

4. The rapid assay of claim 3 wherein, in step (2), the amplification system comprises: PCR Prime STAR HS Premix 25. Mu.L, 10. Mu. Mol/L concentration of each of the upstream and downstream primers 0.25. Mu.L, 1. Mu.L of the template, and water to 50. Mu.L;

5. The rapid detection method according to claim 3, wherein the nucleic acid detection test strip is a colloidal gold test strip with a goat anti-FITC antibody/goat anti-biotin antibody coated on a detection line, a goat anti-mouse antibody coated on a quality control line and a mouse anti-digoxin antibody labeled on colloidal gold, the amplified product is diluted by a detection diluent, and then dripped into a sample adding position of the nucleic acid detection test strip for detection, and the detection diluent is prepared from a citric acid buffer solution containing 0.3mol/L NaCl and 30mmol/L, and has a pH of 7.0.

6. The rapid detection method according to claim 5, wherein the sample is added for 5-8 minutes to obtain a corresponding detection result, and the other time is invalid; in the course of the reading of the result,

when the detection line T line does not develop and the quality control line C line develops, the sample does not contain the sparrow brain component; when the quality control line C does not develop color, the operation is improper or the test paper is failed, and the detection should be repeated.