CN112391451A - Method for pretreating Chinese medicinal preparation - Google Patents
Method for pretreating Chinese medicinal preparation Download PDFInfo
- Publication number
- CN112391451A CN112391451A CN202110078216.XA CN202110078216A CN112391451A CN 112391451 A CN112391451 A CN 112391451A CN 202110078216 A CN202110078216 A CN 202110078216A CN 112391451 A CN112391451 A CN 112391451A
- Authority
- CN
- China
- Prior art keywords
- salmonella
- chinese medicine
- traditional chinese
- solution
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for pretreating a Chinese medicinal preparation. The method comprises the following steps: mixing the oral solid traditional Chinese medicine preparation with the sterile material under the working environment and working conditions which meet the sterility test, and then grinding the mixture into uniformly mixed fine powder to obtain the traditional Chinese medicine pretreatment powder. The traditional Chinese medicine pretreatment powder is used for measuring the salmonella in the traditional Chinese medicine preparation and evaluating the condition of the traditional Chinese medicine preparation polluted by the salmonella according to the measurement result. The salmonella is detected by using a real-time fluorescence PCR method, and the detection of the salmonella by using the real-time fluorescence PCR method comprises the following operation steps: providing a primer and a probe, providing a pre-enrichment strain, extracting salmonella DNA by adopting a thermal cracking method, measuring concentration and purity, carrying out real-time fluorescence PCR amplification, carrying out methodology control, and carrying out result judgment and result expression. The method can quickly and effectively detect the contamination condition of the salmonella in the medicine, particularly the traditional Chinese medicine preparation.
Description
Technical Field
The invention belongs to the technical field of medicines, particularly the technical field of traditional Chinese medicines, and relates to a pretreatment method of a traditional Chinese medicine preparation. The processing material obtained by processing the traditional Chinese medicine preparation by the method can quickly and effectively detect the contamination condition of salmonella in the traditional Chinese medicine preparation.
Background
Salmonella (Salmonella) is a common highly contagious and severely harmful gram-negative bacterium, and Salmonella choleraesuis has been isolated in 1885 by Salmonella et al at the time of cholera epidemic and is therefore designated Salmonella. Salmonella is widely distributed in nature and can survive for 5 months to 2 years in feces, soil, food and water. Feces from people infected with salmonella or carriers contaminate food and can cause food poisoning. According to statistics, in bacterial food poisoning of various countries in the world, food poisoning caused by salmonella is often listed as the head, and the inland areas of China also take salmonella as the head. The salmonella causes the etiological agent to have 3 kinds of invasiveness, endotoxin and enterotoxin, and can clinically cause gastroenteritis, intestinal heat syndrome, bacteremia or septicemia and the like. They can infect many animals including mammals, birds, reptiles, fish, amphibians, and insects in addition to humans. The size of the salmonella is (0.6-0.9) × (1-3) microns, no spores exist, no capsules exist generally, and most of the salmonella is provided with body flagella except for the salmonella pullorum and the salmonella gallinarum. The nutrition requirement is not high, and the separation culture usually adopts an intestinal tract selection identification culture medium. The biochemical reaction has important referential significance for the identification of the bacteria, gelatin is not liquefied, urea is not decomposed, indole is not produced, lactose and sucrose are not fermented, glucose, mannitol and maltose can be fermented, most of the acid and gas are produced, and a few of the acid and gas are produced. VP test is negative, with lysine decarboxylase. The G + C content of the DNA is 50-53%. It is not very resistant to heat and can be killed at 60 deg.C for 15 min, and in 5% carbolic acid, it dies for 5 min. The genus bacteria are divided into 4 sub-genera according to biochemical reaction. Subgenus I is the typical and most common Salmonella species for biochemical reactions; subgenera II and IV are Salmonella atypical for biochemical reactions; subgenus III is Salmonella arizona.
The Chinese medicinal preparation is prepared from Chinese medicinal materials by processing according to a prescribed prescription and preparation process under the guidance of Chinese medicinal theory for preventing and treating diseases. The traditional Chinese medicine is mainly prepared from plant medicines, so that the theory that various medicines are based on grass is provided. The Chinese medicinal materials are mainly derived from natural medicines and processed products thereof, including botanical medicines, animal medicines, mineral medicines and partial chemical and biological products. The salmonella is widely distributed in animals and plants, and the prescription of a large amount of pill-powder minium is often used as the raw powder, so the detection of the salmonella in the traditional Chinese medicine preparation is an important means for evaluating the quality of the medicine and ensuring the safety of the medicine. The general regulation 1107 in the four departments of the 2015 edition is not specified in the microbial limit standard of sterile drugs, and no salmonella (10g) is required to be detected in both the traditional Chinese medicine oral preparation containing the raw material powder and the oral preparation containing the organ extract.
A microbiological culture inspection method for salmonella inspection is determined in the general rule 1106 of the four parts of the Chinese pharmacopoeia 2015 edition, the inspection is carried out by adopting the method, a sample needs to be inoculated into a tryptone soy peptone liquid culture medium with a proper volume, and the mixture is uniformly mixed and cultured for 18-24 hours; then inoculating 0.1ml of the culture into 10ml of RV salmonella enrichment liquid culture medium, and culturing for 18-24 hours; then taking a small amount of the RV salmonella enrichment liquid culture, streaking and inoculating the small amount of RV salmonella enrichment liquid culture on a xylose lysine deoxycholate agar culture medium plate, and culturing for 18-48 hours; if suspected colonies grow, the suspected colonies are selected by an inoculating needle to be inoculated on a trisaccharide iron agar culture medium high-layer inclined plane for inclined plane and high-layer puncture, and are cultured for 18-24 hours or are further identified by other suitable methods. If positive bacteria are identified in the above steps, a suitable identification test is further carried out to confirm whether the bacteria are salmonella. The identification process needs to inoculate the culture into 4 different culture media in sequence, the operation is complicated, the time consumption is long, and only the culture stage needs 72-120 hours. In addition, the detection of the salmonella can be interfered by various external factors, for example, the contaminated salmonella can be damaged or in a dormant state due to the influence of processing steps such as heating, drying and the like in the production process of the medicine, so that the salmonella is difficult to detect; drugs and preservatives may inhibit the proliferation of salmonella, making it difficult to detect; in addition, exogenous pollution is easily introduced by multiple inoculation operations, and the accuracy of a test result is influenced. Although these methods have proven themselves to be reliable, they are laborious and time-consuming, requiring 4-7 days to complete. Therefore, it is difficult to apply the method to a case where the safety of microorganisms in food is generally required to be evaluated promptly and rapidly.
The Real-Time Fluorescence PCR (Real Time Fluorescence PCR, RT-PCR) method is a new detection technology introduced by Applied Biosystems in the United states in 1996, and the Real-Time Fluorescence PCR is a method of adding a fluorescent group into a PCR reaction system, monitoring the whole PCR process in Real Time by using fluorescent signal accumulation, and finally analyzing an unknown template through a standard curve. The advent of fluorescent PCR greatly simplified the detection process and truly achieved absolute quantification. Compared with the conventional PCR, the real-time fluorescence PCR technology has the advantages of stronger specificity, high sensitivity, good repeatability, accurate quantification, high automation degree, full-closed reaction and the like, and becomes an important tool in molecular biology research.
At present, the fluorescence PCR technology is widely applied in the fields of clinical disease diagnosis and curative effect evaluation, animal disease detection, food safety, life science research and the like. In the field of clinical diagnosis of viral diseases such as hepatitis, AIDS, tuberculosis, human papilloma virus detection and the like, the fluorescent PCR technology is widely adopted, and the diagnosis result is quick and accurate; the method is used for detecting the streptococcus pneumoniae and investigating epidemiology, has specific and reliable detection results, has detection sensitivity up to 100fg, has a positive rate of 21 percent for 200 cases of clinical specimens, has a culture method of only 8 percent, has obvious difference, and is quicker and more efficient by the fluorescent PCR method; the real-time fluorescent PCR method of the porcine epidemic diarrhea virus wild strain and the attenuated vaccine strain is established by the Jinsheng and the like, the infection condition and the degree of the porcine epidemic diarrhea virus in a swinery can be directly identified by combining a melting curve, and the rapid and accurate differential diagnosis can be carried out on the wild virus infection and the vaccine immunity of the immune swinery.
Although the fluorescence PCR technology has a certain detection efficiency, no method for effectively detecting salmonella in the traditional Chinese medicine preparation by adopting the real-time fluorescence PCR technology exists at present. Accordingly, a method for rapidly and efficiently detecting salmonella in a chinese medicinal preparation using a real-time fluorescent PCR technique is expected by those skilled in the art.
Disclosure of Invention
The invention aims to provide a method for quickly and effectively detecting salmonella in a traditional Chinese medicine preparation by using a real-time fluorescence PCR method. It has been surprisingly found that the solution of the invention presents excellent technical effects. The present invention has been completed based on such findings.
To this end, a first aspect of the invention provides a method for pre-treating a pharmaceutical formulation, said method comprising the steps of: under the working environment and working conditions which accord with the sterility test, 10 parts by weight of oral solid traditional Chinese medicine preparation, 1.25 parts by weight of glycine and 0.5 part by weight of potassium chloride are mixed, and then are ground into uniformly mixed fine powder to obtain traditional Chinese medicine pretreatment powder; the glycine and potassium chloride were previously subjected to aseptic processing.
The method according to the first aspect of the present invention, wherein the chinese medicinal preparation is selected from: one of stomach invigorating and digestion promoting tablet, JINGUO buccal tablet, YINJIN Capsule, JINSHUIBAO Capsule, WUJIBAIFENG pill, DAHUOLUO pill, and SINIAN powder.
According to the method of the first aspect of the present invention, the Chinese medicinal preparation pretreatment powder obtained is used for the determination of Salmonella therein and the contamination of the Chinese medicinal preparation with Salmonella is evaluated based on the determination result.
The method according to the first aspect of the present invention, wherein the determination of salmonella in the pretreatment powder of traditional Chinese medicine is performed using a real-time fluorescence PCR method, the real-time fluorescence PCR method for detecting salmonella comprises the following steps:
step one, providing a primer and a probe
The sequences of the primers and the probes for detecting the salmonella by the real-time fluorescent PCR method are as follows:
5' end primer, namely upstream primer: 5'-ACAACCTAACTTCTGCGCCA-3' the flow of the air in the air conditioner,
3' end primer, namely downstream primer: 5'-TCAGGTTACCGTGGAGGCTA-3' the flow of the air in the air conditioner,
and (3) probe: 5 '-FAM-CCTTTGTCGTTTTCACCTCGCTGGCTA-BHQ 1-3',
adding proper amount of sterilized deionized water (ddH) into each primer and probe respectively2Diluting O to 10mmol/L, and dissolving with vortex oscillation for 1min, and storing at-20 deg.C;
step two, providing pre-enrichment
Test solution: taking a proper amount of traditional Chinese medicine premix which is equivalent to 10g of traditional Chinese medicine preparation, placing the mixture into a sterile conical flask, adding a TSB culture medium to 200mL, uniformly mixing, and treating for 10min by ultrasonic waves to obtain a test solution;
positive control bacteria liquid of the test sample: adding 1mL of salmonella bacterial liquid with the concentration less than 100cfu/mL into the test solution to obtain a positive control bacterial liquid of the test;
positive control bacterial liquid: adding a TSB culture medium into an aseptic conical flask to 200mL, and then adding 1mL of salmonella bacterial liquid smaller than 100cfu/mL to obtain positive control bacterial liquid;
negative control: TSB medium was used as negative control;
the solutions are respectively placed in a constant-temperature incubator at 36 ℃ for overnight culture for 16 hours and then used as experimental bacteria liquid for subsequent DNA extraction;
step three, extracting salmonella DNA by adopting a thermal cracking method
Respectively taking 1mL of the enriched experimental bacteria liquid, namely the test sample solution, the test sample positive control bacteria liquid, the positive control bacteria liquid and the negative control, placing the enriched experimental bacteria liquid, namely the test sample solution, the test sample positive control bacteria liquid, the positive control bacteria liquid and the negative control into a 1.5mL sterile centrifuge tube, centrifuging the mixture at the speed of 12000rpm for 2min, discarding the supernatant, resuspending the thallus precipitate in 200 mu L of sterile water, heating the mixture in a water bath kettle at the temperature of 95 ℃ for 10min, centrifuging the mixture at the speed of 12000rpm for 5min, and taking the supernatant as a PCR reaction template DNA solution; after the concentration and the purity are detected, storing at-20 ℃ for later use;
step four, determination of DNA concentration and purity
Measuring the concentration and purity of the DNA by an ultraviolet spectrophotometer;
step five, real-time fluorescence PCR amplification
The total volume of the reaction system is 25 mu L, and PCR reaction solution is prepared according to the following proportion: 1 muL of template DNA solution (for example, 10 ng-100 ng of DNA is contained in the template DNA solution), 0.2 muL of Taq enzyme (for example, 5U/. mu.L), 5 muL of 10 XPCR Buffer, 1 muL of dNTP Mix, 1 muL of upstream primer, 1 muL of downstream primer, 0.5 muL of probe, and a proper amount of supplementary solution 1 mu L, ddH2O to 25 muL,
and (3) PCR reaction conditions:
i. 95 ℃: pre-denaturation for 3 min;
ii. 95 ℃: denaturation for 10 s;
iii, 64 ℃: annealing for 30s, and collecting fluorescence; the above ii and iii steps are carried out for 40 times of circulation;
iv, stopping;
setting positive control, positive control of the test sample, negative control and blank control simultaneously in the detection process;
calculating the number N of total PCR reaction tubes, preparing N +1 parts of PCR reaction solution into a 1.5ml sterile centrifuge tube according to the reaction system, fully oscillating and uniformly mixing, centrifuging at low speed for 10s, subpackaging into PCR 8 reaction tubes, adding a positive control DNA extracting solution into a positive control tube, adding a test sample DNA extracting solution into a test sample tube, adding a test sample positive control DNA extracting solution into a test sample positive control tube, adding a negative control extracting solution into a negative control tube, adding equal volume of water into a blank control tube to replace the DNA extracting solution, and covering a reaction tube cover; fully and uniformly mixing the liquid in the PCR reaction tube, removing bubbles in the reaction tube, and centrifuging at a low speed for 30s for later use;
opening the fluorescent quantitative PCR instrument, putting the fluorescent quantitative PCR instrument into the PCR reaction tube, closing the cover, and operating the program; after the program operation is finished, analyzing an experimental result, determining a threshold line, and outputting a report of parameters including FAM fluorescence signals and Ct values;
step six, methodology control:
the result is considered invalid when one of the following conditions is not satisfied:
a. blank control: no FAM fluorescent signal is detected,
b. negative control: no FAM fluorescent signal is detected,
c. positive control: detecting FAM fluorescence signals, wherein a typical amplification curve appears in an FAM channel, the Ct value is less than or equal to 35.0,
d. positive control of test article: detecting FAM fluorescent signals, wherein a typical amplification curve appears in an FAM channel, and the Ct value is less than or equal to 35.0;
step seven, result judgment and result expression:
a. if the Ct value is less than or equal to 35.0, the tested sample is judged to be positive, namely the traditional Chinese medicine preparation is positive, and the result is expressed as 'salmonella is detected';
b. if the Ct value is more than or equal to 40.0, the sample to be detected is judged to be negative, namely the traditional Chinese medicine preparation is negative, and the result is expressed as 'no salmonella' detected;
c. if the Ct value is more than 35.0 and less than 40.0, repeating the test once, and if the Ct value is still more than 35.0 and less than 40.0 after the re-amplification, determining that the sample to be detected is the Chinese medicinal preparation is suspicious and is expressed as 'salmonella suspicious'.
The method according to the first aspect of the present invention, wherein the operation of measuring the DNA concentration and purity with an ultraviolet spectrophotometer is as follows: after the ultramicro ultraviolet/visible spectrophotometer is started, entering a detection interface, adding 2ul of blank solution into a detection hole, clicking reference detection to adjust zero, and sucking liquid in the detection hole by using absorbent paper after the zero is adjusted; then adding 2ul of sample solution to be detected, clicking the sample for detection, reading and recording the obtained DNA concentration value and A280/A260 ratio, sucking the liquid in the detection hole by using absorbent paper, and repeating the operation method to continuously detect the next sample.
According to the method of the first aspect of the invention, the supplementation solution is an aqueous solution comprising 2 μmol/L sodium selenite and 6mg/ml fructose.
According to the method of the first aspect of the present invention, the template DNA solution in the PCR reaction solution contains 10ng to 100ng of DNA per 1. mu.L.
According to the method of the first aspect of the present invention, the concentration of the Taq enzyme is 5U/. mu.L.
According to the method of the first aspect of the present invention, the four deoxynucleotides contained in the dNTP Mix are each contained at a concentration of 10 mM.
The method according to the first aspect of the present invention, wherein the concentration of the DNA detected is 1.5X 10-3μg/mL~1.5×103Linear regression correlation coefficient R in mu g/mL range2Greater than 0.999.
According to the method of the first aspect of the present invention, salmonella is detected with a specificity of more than 99% without interference from other bacterial genes.
According to the method of the first aspect of the present invention, the sensitivity of detecting salmonella in a 10g sample can reach 1 cfu.
The method according to the first aspect of the invention, which detects salmonella in a 10g sample, has a sensitivity of less than 1 cfu.
Further, the second aspect of the present invention provides a PCR reaction solution, which contains, per 25. mu.L: 1 mu L of template DNA solution (containing 10 ng-100 ng of DNA), 0.2 mu L of Taq enzyme (5U/mu L), 5 mu L of 10 XPCR Buffer, 1 mu L of dNTP Mix, 1 mu L of upstream primer, 1 mu L of downstream primer, 0.5 mu L of probe, and proper amount of 1 mu L, ddH2O aqueous solution containing 2 mu mol/L sodium selenite and 6mg/ml fructose to 25 mu L.
Further, the third aspect of the invention provides a traditional Chinese medicine pretreatment powder, which comprises an oral solid traditional Chinese medicine preparation, glycine and potassium chloride, and is prepared by the following steps: under the working environment and working conditions which accord with the sterility test, 10 parts by weight of oral solid traditional Chinese medicine preparation, 1.25 parts by weight of glycine and 0.5 part by weight of potassium chloride are mixed, and then are ground into uniformly mixed fine powder to obtain traditional Chinese medicine pretreatment powder; the glycine and potassium chloride were previously subjected to aseptic processing.
The traditional Chinese medicine pretreatment powder according to the third aspect of the invention, wherein the traditional Chinese medicine preparation is selected from the group consisting of: one of stomach invigorating and digestion promoting tablet, JINGUO buccal tablet, YINJIN Capsule, JINSHUIBAO Capsule, WUJIBAIFENG pill, DAHUOLUO pill, and SINIAN powder.
The fourth aspect of the invention provides the use of the traditional Chinese medicine pretreatment powder of any one of the third aspect of the invention in the preparation of a test article for detecting the salmonella contamination condition of a traditional Chinese medicine preparation; wherein the method for detecting the salmonella contamination condition of the traditional Chinese medicine preparation adopts the real-time fluorescence PCR method of the first aspect of the invention.
In the method steps of the present invention, although the specific steps described therein differ in some detail or in language from the steps described in the examples of the detailed description section below, those skilled in the art can, nevertheless, fully generalize the above-described method steps in light of the detailed disclosure of the present invention.
Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
The present invention exhibits the technical effects as described in the present specification.
Drawings
FIG. 1: and (5) diluting the detection result by 10 times of gradient of the salmonella DNA solution.
FIG. 2: the specific detection result shows that m is 19 salmonella strains, n is 43 non-salmonella strains and negative control in the figure.
FIG. 3: the detection results of 18 batches of Chinese medicinal preparations are shown in the figure, wherein X is a positive control, and Y is a positive control of a test sample.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The invention provides a method for detecting salmonella in a traditional Chinese medicine preparation by a real-time fluorescence PCR method, which provides reference for realizing rapid detection and identification of the salmonella in the traditional Chinese medicine preparation. The basic principle and the operation process of the method are that the traditional Chinese medicine preparation is added into a trypticase soy peptone liquid culture medium for enrichment culture, the enrichment liquid is taken to extract genome DNA in a sample, the DNA is taken as a template, species-specific primers and probes are utilized to carry out real-time fluorescence PCR amplification detection, and positive, negative and blank controls are set simultaneously. And judging whether the sample contains salmonella or not according to the Ct value of the amplification. The glycine and potassium chloride used in the examples of the present invention were previously subjected to aseptic processing, especially uncontaminated salmonella.
Preparation example 1: preparation of traditional Chinese medicine pretreatment powder
Under the working environment and working conditions which accord with the sterility test, one of the 18 batches of the traditional Chinese medicine preparations in the table 2 is taken respectively, and the following traditional Chinese medicine preparations are adopted: glycine: potassium chloride is mixed according to a weight ratio of 10: 1.25: 0.5, then grinding the mixture into uniformly mixed fine powder to obtain the traditional Chinese medicine pretreatment powder of different products and different batches.
Preparation example 2: preparation of traditional Chinese medicine pretreatment powder
Under the working environment and working conditions which accord with the sterility test, one of the 18 batches of the traditional Chinese medicine preparations in the table 2 is taken respectively, and the following traditional Chinese medicine preparations are adopted: glycine is mixed in a weight ratio of 10: 1.25, then grinding the mixture into uniformly mixed fine powder to obtain the traditional Chinese medicine pretreatment powder of different products and different batches.
Preparation example 3: preparation of traditional Chinese medicine pretreatment powder
Under the working environment and working conditions which accord with the sterility test, one of the 18 batches of the traditional Chinese medicine preparations in the table 2 is taken respectively, and the following traditional Chinese medicine preparations are adopted: potassium chloride is mixed according to a weight ratio of 10: 0.5, then grinding the mixture into uniformly mixed fine powder to obtain the traditional Chinese medicine pretreatment powder of different products and different batches.
Preparation example 4: preparation of traditional Chinese medicine pretreatment powder
Under the working environment and working conditions which meet the sterility test, one of the 18 batches of the traditional Chinese medicine preparations shown in the table 2 is respectively taken and ground into uniform fine powder, so as to obtain the traditional Chinese medicine pretreatment powder of different products in different batches.
Referring to the preparation method of the test solution in "2, providing pre-enrichment" of example 1, a proper amount of each of the premixes of the traditional Chinese medicines of preparation examples 1 to 4, which contain 10g of the traditional Chinese medicine preparation, was taken, put into a sterile conical flask, added with the TSB medium to 200mL, mixed well, and treated with ultrasonic waves for 10min to obtain the test solution. It has been found that, surprisingly, after the ultrasonic homogenization treatment: the sample solutions obtained from all of the pre-treated powders of preparations 2-4 exhibited substantial non-digestible foam (it is expected that 50-120 ml of foam could be observed in the erlenmeyer flask, e.g., about 95ml of foam in the sample solution obtained from the tetrad of preparation 4); whereas the sample solution obtained from the whole pretreatment powder of preparation example 1 showed no foam or only a slight amount of foam scattered on floating (it can be estimated that less than 2ml of foam was observed in a conical flask, for example, the sample solution obtained from preparation example 1 by treating tetrakiss powder showed no foam). These foams are extremely disadvantageous for the subsequent measurement, especially because the amount of microorganisms adhered to the foam part is 0.5 to 2 orders of magnitude larger than the amount of microorganisms in the main solution of the test solution due to the difference in adsorption capacity, which was found in the additional experiments of the present inventors. In this sense, pretreatment of the traditional Chinese medicine preparation with glycine and potassium chloride in preparation example 1 is beneficial to measurement of salmonella in the traditional Chinese medicine preparation.
Example 1: real-time fluorescence PCR method for detecting salmonella in traditional Chinese medicine preparation
The premix of the present example was prepared by the method of preparation example 1, unless otherwise specified.
1. Providing primers and probes
The sequences of the primers and the probes for detecting the salmonella by the real-time fluorescent PCR method are as follows:
5' end primer (also called upstream primer): 5'-ACAACCTAACTTCTGCGCCA-3' the flow of the air in the air conditioner,
3' end primer (also called downstream primer): 5'-TCAGGTTACCGTGGAGGCTA-3' the flow of the air in the air conditioner,
and (3) probe: 5 '-FAM-CCTTTGTCGTTTTCACCTCGCTGGCTA-BHQ 1-3',
adding proper amount of sterilized deionized water (ddH) into each primer and probe respectively2O) diluting to 10mmol/L, and dissolving by vortex oscillation for 1min, and storing at-20 ℃ for later use;
the sequences of these primers and probes can be easily synthesized by a known primer synthesis method, or can be obtained commercially, for example, by entrusting commercial primer synthesis.
In addition, some commonly used media and reagents are provided (main listed):
physiological saline, Trypticase Soytone Broth (TSB), Taq DNA Polymerase, dNTP Mix, 10 XPCR Buffer, ddH2And O. For example, if used, physiological saline (available from Shigaku chemical Co., Ltd.), trypticase Soy peptone broth (TSB) (available from Beijing Luqiao Bio Inc.), Taq DNA Polymerase (5U/. mu.L, Taq enzyme) (available from Kang Shijiki Biotech Co., Ltd.), dNTP Mix (10 mM each, available from Kangshui Shiji Biotech Co., Ltd.), 10 XPCR Buffer (available from Kangshui Shiji Biotech Co., Ltd.), 6 XPDNA Loading Buffer (available from Kangshui Shiji Biotech Co., Ltd.), 10000 Xgold View (available from Beijing Lei Toji Biotech Co., Ltd.), 100bp Ladder (available from Kangshui Shiji Biotech Co., Ltd.), 50 XTAE Tris-acetate electrophoresis Buffer (available from Beijing Lei Toji Biotech Co., Ltd.), agarose (Spanish, available from Kangshui Shiji Biotechnology Co., Ltd.).
In addition, there are provided some commonly used instruments and equipments, a fluorescence quantitative PCR instrument CFX96Touch (U.S. Bio-Rad Belle), an ultra-micro UV/visible spectrophotometer Spectra Max Quick Drop (Meigu molecule), a desk-top high speed refrigerated centrifuge 5424R (El Bend Ltd.), a high pressure steam sterilizer YXQ-LS-50 SII (Shanghai Bo Xun Shi medical instruments Co., Ltd.), a microwave oven Grace D8523CTL-2W (Shundland microwave oven electric Co., Ltd.), an electronic balance Sartorius T601-L (Beijing Saedodes instruments systems Co., Ltd.), a biosafety cabinet Heal Force-900C (U.S. Sammer Feill science Co., Ltd.), a bacteria incubator PYX-DHS (Shanghai Tan medical instruments Co., Ltd.), a constant temperature water bath, a vortex shaker, a palm centrifuge, a micropipeter, and the like.
2. Enrichment before providing
Test solution: taking a proper amount of traditional Chinese medicine premix which is equivalent to 10g of traditional Chinese medicine preparation, placing the mixture into a sterile conical flask, adding a TSB culture medium to 200mL, uniformly mixing, and treating for 10min by ultrasonic waves to obtain a test solution;
positive control bacteria liquid of the test sample: adding 1mL of salmonella bacterial liquid with the concentration less than 100cfu/mL into the test solution to obtain a positive control bacterial liquid of the test;
positive control bacterial liquid: adding a TSB culture medium into an aseptic conical flask to 200mL, and then adding 1mL of salmonella bacterial liquid smaller than 100cfu/mL to obtain positive control bacterial liquid;
negative control: TSB medium was used as negative control;
the solutions are respectively placed in a constant-temperature incubator at 36 ℃ for overnight culture (16h) and then used as experimental bacteria liquid for subsequent DNA extraction;
3. extraction of salmonella DNA by thermal cracking
Respectively taking 1mL of the enriched experimental bacteria liquid, namely the test sample solution, the test sample positive control bacteria liquid, the positive control bacteria liquid and the negative control, placing the enriched experimental bacteria liquid, namely the test sample solution, the test sample positive control bacteria liquid, the positive control bacteria liquid and the negative control into a 1.5mL sterile centrifuge tube, centrifuging the mixture at the speed of 12000rpm for 2min, discarding the supernatant, resuspending the thallus precipitate in 200 mu L of sterile water, heating the mixture in a water bath kettle at the temperature of 95 ℃ for 10min, centrifuging the mixture at the speed of 12000rpm for 5min, and taking the supernatant as a PCR reaction template DNA solution; after the concentration and the purity are detected, storing at-20 ℃ for later use;
4. determination of DNA concentration and purity
Measuring the concentration and purity of DNA with an ultraviolet spectrophotometer (the conventional operation method comprises the steps of starting up an ultramicro ultraviolet/visible spectrophotometer, entering a detection interface, adding 2ul of blank solution into a detection hole, clicking reference detection to zero, sucking the liquid in the detection hole with absorbent paper after zero setting, then adding 2ul of sample solution to be detected, clicking the sample to detect, reading and recording the obtained concentration value of the DNA and the A280/A260 ratio, sucking the liquid in the detection hole with the absorbent paper, and repeating the operation method to continuously detect the next sample);
5. real-time fluorescent PCR amplification
The total volume of the reaction system is 25 mu L, and PCR reaction solution is prepared according to the following proportion: 1 muL of template DNA solution (containing 10 ng-100 ng of DNA), 0.2 muL of Taq enzyme (5U/muL), 5 muL of 10 XPCR Buffer, 1 muL of dNTP Mix, 1 muL of upstream primer, 1 muL of downstream primer, 0.5 muL of probe and a proper amount of supplementary solution 1 mu L, ddH2O to 25 muL;
and (3) PCR reaction conditions:
i. 95 ℃: pre-denaturation for 3 min;
ii. 95 ℃: denaturation for 10 s;
iii, 64 ℃: annealing for 30s, and collecting fluorescence; the above ii and iii steps are carried out for 40 times of circulation;
iv, stopping;
setting positive control, positive control of the test sample, negative control and blank control simultaneously in the detection process;
the supplementing liquid is an aqueous solution containing 2 mu mol/L sodium selenite and 6mg/ml fructose;
calculating the number N of total PCR reaction tubes, preparing N +1 parts of PCR reaction solution into a 1.5ml sterile centrifuge tube according to the reaction system, fully oscillating and uniformly mixing, centrifuging at low speed for 10s, subpackaging into PCR 8 reaction tubes, adding a positive control DNA extracting solution into a positive control tube, adding a test sample DNA extracting solution into a test sample tube, adding a test sample positive control DNA extracting solution into a test sample positive control tube, adding a negative control extracting solution into a negative control tube, adding equal volume of water into a blank control tube to replace the DNA extracting solution, and covering a reaction tube cover; and (3) fully and uniformly mixing the liquid in the PCR reaction tube, removing air bubbles in the reaction tube, and centrifuging at a low speed for 30s for later use.
Opening the fluorescent quantitative PCR instrument, putting the fluorescent quantitative PCR instrument into the PCR reaction tube, closing the cover, and operating the program; after the program operation is finished, analyzing an experimental result, determining a threshold line, and outputting a report of parameters including FAM fluorescence signals and Ct values;
6. and (3) controlling the methodology:
the result is considered invalid when one of the following conditions is not satisfied:
a. blank control: no FAM fluorescent signal is detected,
b. negative control: no FAM fluorescent signal is detected,
c. positive control: detecting FAM fluorescence signals, wherein a typical amplification curve appears in an FAM channel, the Ct value is less than or equal to 35.0,
d. positive control of test article: detecting FAM fluorescent signals, wherein a typical amplification curve appears in an FAM channel, and the Ct value is less than or equal to 35.0;
7. result determination and result expression:
a. if the Ct value is less than or equal to 35.0, the tested sample is judged to be positive, namely the traditional Chinese medicine preparation is positive, and the result is expressed as 'salmonella is detected';
b. if the Ct value is more than or equal to 40.0, the sample to be detected is judged to be negative, namely the traditional Chinese medicine preparation is negative, and the result is expressed as 'no salmonella' detected;
c. if the Ct value is more than 35.0 and less than 40.0, repeating the test once, and if the Ct value is still more than 35.0 and less than 40.0 after the re-amplification, determining that the sample to be detected is the Chinese medicinal preparation is suspicious and is expressed as 'salmonella suspicious'.
Example 2: methodological evaluation of real-time fluorescent PCR method
In order to prove that the method established in the research is suitable for the rapid detection of the salmonella in the traditional Chinese medicine preparation, the established method needs to be subjected to corresponding methodology evaluation research, including the evaluation of the detection linear range, the detection specificity, the detection sensitivity, the actual application effect of the method and the like.
In example 2, the methodology was evaluated for the real-time fluorescent PCR method described in example 1.
1. Use of strains
This example 2 used 19 strains of salmonella and 43 strains of non-salmonella of the names and sources shown in table 1 below.
Table 1:
2. detecting linear range
2mL of positive control bacterial liquid of Salmonella paratyphi B is taken, DNA is extracted by the DNA extraction method described in example 1, and then the concentration and quality of nucleic acid are detected by an ultra-micro ultraviolet-visible spectrophotometer. The DNA solution was used as a mother solution, and 10-fold gradient dilution was performed with sterile water to dilute 9 gradients, and 2. mu.L of each of the above DNA solutions was collected, and each concentration was repeated in parallel, and the detection was performed by the detection method described in example 1. And analyzing the detection result, performing linear regression analysis by taking the CT value as a vertical coordinate and the DNA concentration logarithm value as a horizontal coordinate, and detecting the linear range by using the evaluation method.
As a result: after extracting DNA from positive control bacteria liquidThe nucleic acid concentration detected by an ultramicro ultraviolet-visible spectrophotometer is 1526 mug/mL, and the A260/280 ratio is 2.096. The result of real-time fluorescent quantitative PCR detection after 10-fold gradient dilution of the DNA solution is shown in FIG. 1A, and the average CT value of each dilution level is: stock solution (13.12), 10-1(16.24)、10-2(19.635)、10-3(23.095)、10-4(26.41)、10-5(29.645)、10-6(32.675)，10-7Only one parallel had specific amplification, with a CT value of 35.47. The results of linear regression analysis using CT values as ordinate and log values of nucleic acid concentration as abscissa are shown in FIG. 1B.
As can be seen from FIG. 1A, the detection method can accurately detect 10-6Dilution grade, i.e. 1.5X 10-3Mu g/mL DNA solution, and the standard deviation between the parallel treatments of each dilution level is less than 1.31 percent, which shows that the method has very low detection limit and better repeatability. FIG. 1B shows that the amplification efficiency of the PCR reaction was 101.1%, and the linear regression correlation coefficient R between the CT value and the logarithm of the nucleic acid concentration2Is 1.0, which shows that the method can accurately detect the DNA concentration at 1.5X 10-3μg/mL~1.5×103Samples in the μ g/mL range and the log values of the nucleic acid concentration in this concentration range are linearly related to the CT value.
The results of the linear test show that the methodology of example 1 is excellent.
3. Detection specificity
19 Salmonella strains and 43 non-Salmonella strains listed in the above Table were inoculated into 200mL of TSB, respectively, and cultured in a 36 ℃ incubator for 24 hours, as described in example 1, and then DNA was extracted by the DNA extraction method, followed by detection to examine the detection specificity of the analysis method.
The detection results of 19 salmonella strains and 43 non-salmonella strains are shown in figure 2, and it can be seen that 19 salmonella strains have typical S-shaped fluorescence increase curves, and CT values are all less than 22, while 43 non-salmonella strains have no fluorescence increase curves, which indicates that the primer probe adopted in the method can specifically detect salmonella without being interfered by other bacterial genes, and the specificity is 100%.
4. Method sensitivity
Taking 1mL of overnight cultured salmonella paratyphi B bacterial liquid, carrying out 10-fold gradient dilution by using sterile normal saline, diluting 10 concentrations, respectively taking 1mL of each dilution stage, coating 2 TSA plates (0.5 mL/dish), placing the plates in a constant-temperature incubator at 36 ℃ for 24 hours, counting the bacterial colony number of each plate, and calculating the bacterial liquid concentration. Meanwhile, 1mL of the stock solution and 1mL of each dilution-grade bacterial solution were inoculated into 200mL of TSB medium, and the stock solution and each dilution-grade bacterial solution were cultured overnight (16 hours) in a 36 ℃ incubator, respectively, and DNA was extracted as described in example 1, followed by detection to evaluate the detection sensitivity of the method.
As a result: the overnight inoculum concentration was 4.6X 10 by plate count9cfu/mL. DNA detection by taking overnight-cultured bacterial liquid of each dilution grade is found that all dilution grades (including 10 th dilution grade, the concentration is 0.46cfu/mL) are amplified, and CT values are all less than 20, which indicates that the limit of the method for detecting salmonella in a sample is 0.46cfu (or single cfu) theoretically, namely when a test sample is tested, only 1cfu of salmonella in 10g of the sample can be detected by the method, so that the method has high detection sensitivity. Meanwhile, the enrichment condition that TSB is used as a culture medium and cultured at the constant temperature of 36 ℃ overnight (16h) is also shown to be enough to meet the subsequent detection requirement.
The result of the embodiment 2 shows that the method of the embodiment 1 of the invention has excellent methodological characteristics and can completely meet the requirement of measuring the salmonella in the traditional Chinese medicine preparation.
Example 3: the invention uses the real-time fluorescence PCR method to detect the salmonella in the traditional Chinese medicine preparation
As described in example 2, the method of example 1 of the present invention has excellent methodological characteristics, and can completely satisfy the requirement of salmonella in the traditional Chinese medicine preparation. Based on these works, in example 3, 18 batches of commercially available traditional Chinese medicine preparations (including 8 tablets, 6 pills, 3 capsules, 1 powder and details shown in table 2) were used as a study object, and a test solution was prepared by the method of example 1, using a traditional Chinese medicine preparation that requires salmonella test according to the regulations of the "chinese pharmacopoeia" as a test object, and the test was performed according to the method established in example 1 and the "1106 non-sterile product microorganism limit test" of the four general rules of the "chinese pharmacopoeia" 2015 edition: the methods in section "control bacteria examination method" were examined simultaneously to evaluate the actual application effects of the methods.
As a result: the real-time fluorescent quantitative PCR detection results of the 18 batches of the traditional Chinese medicine preparations are shown in a figure 3, the positive control bacterial liquid and all the 18 batches of the positive control bacterial liquid of the test sample have typical S-shaped fluorescent growth curves, and the result shows that the 18 batches of the samples have no inhibition effect on the growth of the salmonella paratyphi B; meanwhile, all the test solutions and the negative control have no fluorescence increase curve, which indicates that 18 batches of samples have no salmonella pollution. The detection results are consistent with the detection results according to the pharmacopoeia method (see table 2), which shows that the method established in the embodiment 2 of the invention can meet the requirement of detecting salmonella in the traditional Chinese medicine preparation and can be used as an effective supplement of the pharmacopoeia method.
Table 2: results of two methods for detecting 18 batches of Chinese medicinal preparations are compared
In the table, -: no salmonella was detected, +: detecting salmonella; when a plurality of batches are represented by the same symbol, the result representing the batches is the same as the symbol.
As described above, according to the results of examples 1 to 3 of the present invention, it can be seen that the method of the present invention has an excellent effect of examining Salmonella in a Chinese medicinal preparation. In the Chinese pharmacopoeia 2015, a bacterial culture inspection method is adopted to inspect salmonella, while in the general rules of the four parts of the pharmacopoeia 2020, a molecular biology inspection method is added, but mainly aims at species identification and bacterial identification of traditional Chinese medicines and the like, and does not relate to inspection of control bacteria, and a PCR detection method for salmonella in a traditional Chinese medicine preparation is currently reported rarely. The research adopts one-time enrichment and thermal cracking method to extract DNA, then uses a real-time fluorescence quantitative PCR method to detect salmonella in the traditional Chinese medicine preparation, the detection can be finished within 18h at the fastest speed, and the detection linear range of using the B paratyphoid salmonella DNA solution as a template is 1.5 multiplied by 10-3μg/mL~1.5×103Mu g/mL, can specifically detect salmonella, has the sensitivity of 1cfu/10g of test sample, and adopts the method to detect 18 batches of samplesThe results of the medicinal preparation are consistent with the method of Chinese pharmacopoeia. The limit requirement of the non-sterile medicine microorganism limit standard on salmonella is that salmonella cannot be detected (10g), so the sensitivity of the detection method must reach a single cfu, the method established by the research can meet the requirement, and has the advantages of shorter detection period and the like, can be used as effective supplement of a method of pharmacopoeia and is used for rapid detection of salmonella in a Chinese medicinal preparation.
In a supplementary example (which is referred to herein as example 1A), reference is made to the method of example 1, except that the supplementary liquid used is not supplemented with sodium selenite; as a result: the overnight inoculum concentration was 7.4X 10 by plate count9cfu/mL, DNA extraction and detection are carried out on the bacteria liquid obtained by overnight culture of each dilution level, all the 1 st to 9 th dilution levels (including the 9 th dilution level, the concentration is 7.4cfu/mL) are amplified, and the CT value is less than 26, but the 10 th dilution level is not detected, which indicates that the limit of the method for theoretically detecting the salmonella in the sample is 7.4 cfu. In a supplementary example (which is referred to herein as example 1B), the process of example 1 is referenced, except that the supplemental liquid used is not fructose; as a result: the overnight inoculum concentration was 3.9X 10 by plate count9cfu/mL, DNA extraction and detection are carried out on the bacteria liquid obtained by overnight culture of each dilution grade, all the 1 st to 9 th dilution grades (including the 9 th dilution grade, the concentration is 3.9cfu/mL) are amplified, and the CT value is less than 19, but the 10 th dilution grade is not detected, which indicates that the limit of the method for theoretically detecting the salmonella in the sample is 3.9 cfu. In a supplementary example (which is referred to herein as example 1C), reference is made to the method of example 1, except that the supplementation solution is replaced with an equal volume of ddH2O, i.e. no supplementation solution is added; as a result: the overnight inoculum concentration was 5.3X 10 by plate count9cfu/mL, DNA extraction and detection are carried out on the bacteria liquid obtained by overnight culture of each dilution grade, all the 1 st to 9 th dilution grades (including the 9 th dilution grade, the concentration is 5.3cfu/mL) are amplified, and the CT value is less than 28, but the 10 th dilution grade is not detected, which shows that the limit of the method for theoretically detecting the salmonella in the sample is 5.3 cfu.It can be seen that the sensitivity of the method according to example 1 was reduced by as much as an order of magnitude when no sodium selenite and/or no fructose was added to the supplementation solution, indicating that the simultaneous addition of sodium selenite and fructose in the supplementation solution is necessary.
The traditional Chinese medicine preparation has complex formula and various components, and a plurality of components can inhibit the growth of salmonella. Therefore, as in the pharmacopeia detection method, the setting of the positive control and the negative control is very important for controlling the quality of the detection result, and in order to avoid the occurrence of false negative results, the positive control and the positive control of the test sample need to be set at the same time. Once the positive control result is positive and the positive control result of the test sample is negative, the obtained result is probably false negative, which indicates that the preparation can inhibit the proliferation of the salmonella, the methodological verification is carried out when the positive control result is negative, and if necessary, the growth inhibition effect of the preparation on the salmonella can be eliminated by adopting a membrane filtration method for washing and the like, and then the enrichment detection is carried out. The invention detects 18 batches of Chinese medicinal preparations, and no obvious inhibition effect of the medicament on the growth of salmonella is found, which indicates that the method is reliable. In addition, the situation of false negative in the detection process can be avoided by adding an internal reference control in the detection.
The research establishes a real-time fluorescent quantitative PCR detection method for salmonella in the traditional Chinese medicine preparation, and the specific primer probe is adopted, so that whether the sample pollutes the salmonella can be specifically detected, and the interference of other microorganisms and bacteriostatic components in the sample is avoided, thereby omitting the pure culture step; meanwhile, by utilizing the characteristics of high sensitivity, high automation degree, quick reaction and the like of the real-time fluorescent quantitative PCR technology, the sample can be used for detection after overnight enrichment, and the detection period is shortened to be within 24 hours; in addition, because the passage steps are reduced and the PCR reaction is carried out in a closed system, cross contamination with the outside is not easy to generate. Therefore, the establishment of the real-time fluorescence PCR detection method for salmonella in the traditional Chinese medicine preparation can overcome many defects in the traditional bacteria culture detection method, and the method can be used as a quick detection method, save time and labor cost in drug microorganism detection, and can be used for salmonella detection in the fields of traditional Chinese medicine decoction pieces, food and the like after being properly adjusted.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> institute for drug testing and detection in Jiangxi province
<120> method for pretreating Chinese medicinal preparation
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 27
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cctttgtcgt tttcacctcg ctggcta 27
Claims (10)
1. A method of pretreating a traditional Chinese medicine preparation, the method comprising the steps of: under the working environment and working conditions which accord with the sterility test, 10 parts by weight of oral solid traditional Chinese medicine preparation, 1.25 parts by weight of glycine and 0.5 part by weight of potassium chloride are mixed, and then are ground into uniformly mixed fine powder to obtain traditional Chinese medicine pretreatment powder; the glycine and potassium chloride were previously subjected to aseptic processing.
2. The method of claim 1, wherein the chinese medicinal formulation is selected from the group consisting of: one of stomach invigorating and digestion promoting tablet, JINGUO buccal tablet, YINJIN Capsule, JINSHUIBAO Capsule, WUJIBAIFENG pill, DAHUOLUO pill, and SINIAN powder.
3. The method as set forth in claim 1, wherein the pre-treated powder of chinese traditional medicine obtained is used for the determination of salmonella therein and the contamination of the chinese traditional medicine preparation with salmonella is evaluated based on the determination result.
4. The method as claimed in claim 3, wherein the determination of Salmonella in the pretreatment powder of Chinese medicine is performed by using a real-time fluorescence PCR method, the real-time fluorescence PCR method for detecting Salmonella comprises the following steps:
step one, providing a primer and a probe
The sequences of the primers and the probes for detecting the salmonella by the real-time fluorescent PCR method are as follows:
5' end primer, namely upstream primer: 5'-ACAACCTAACTTCTGCGCCA-3' the flow of the air in the air conditioner,
3' end primer, namely downstream primer: 5'-TCAGGTTACCGTGGAGGCTA-3' the flow of the air in the air conditioner,
and (3) probe: 5 '-FAM-CCTTTGTCGTTTTCACCTCGCTGGCTA-BHQ 1-3',
adding proper amount of sterilized deionized water (ddH) into each primer and probe respectively2Diluting O to 10mmol/L, and dissolving with vortex oscillation for 1min, and storing at-20 deg.C;
step two, providing pre-enrichment
Test solution: taking a proper amount of traditional Chinese medicine premix which is equivalent to 10g of traditional Chinese medicine preparation, placing the mixture into a sterile conical flask, adding a TSB culture medium to 200mL, uniformly mixing, and treating for 10min by ultrasonic waves to obtain a test solution;
positive control bacteria liquid of the test sample: adding 1mL of salmonella bacterial liquid with the concentration less than 100cfu/mL into the test solution to obtain a positive control bacterial liquid of the test;
positive control bacterial liquid: adding a TSB culture medium into an aseptic conical flask to 200mL, and then adding 1mL of salmonella bacterial liquid smaller than 100cfu/mL to obtain positive control bacterial liquid;
negative control: TSB medium was used as negative control;
the solutions are respectively placed in a constant-temperature incubator at 36 ℃ for overnight culture for 16 hours and then used as experimental bacteria liquid for subsequent DNA extraction;
step three, extracting salmonella DNA by adopting a thermal cracking method
Respectively taking 1mL of the enriched experimental bacteria liquid, namely the test sample solution, the test sample positive control bacteria liquid, the positive control bacteria liquid and the negative control, placing the enriched experimental bacteria liquid, namely the test sample solution, the test sample positive control bacteria liquid, the positive control bacteria liquid and the negative control into a 1.5mL sterile centrifuge tube, centrifuging the mixture at the speed of 12000rpm for 2min, discarding the supernatant, resuspending the thallus precipitate in 200 mu L of sterile water, heating the mixture in a water bath kettle at the temperature of 95 ℃ for 10min, centrifuging the mixture at the speed of 12000rpm for 5min, and taking the supernatant as a PCR reaction template DNA solution; after the concentration and the purity are detected, storing at-20 ℃ for later use;
step four, determination of DNA concentration and purity
Measuring the concentration and purity of the DNA by an ultraviolet spectrophotometer;
step five, real-time fluorescence PCR amplification
The total volume of the reaction system is 25 mu L, and PCR reaction solution is prepared according to the following proportion: template DNA solution 1 mu L, Taq enzyme 0.2 mu L, 10 XPCR Buffer 5 mu L, dNTP Mix 1 mu L, upstream primer 1 mu L, downstream primer 1 mu L, probe 0.5 mu L, supplementary liquid 1 mu L, ddH2O in proper amount to 25 mu L,
and (3) PCR reaction conditions:
i. 95 ℃: pre-denaturation for 3 min;
ii. 95 ℃: denaturation for 10 s;
iii, 64 ℃: annealing for 30s, and collecting fluorescence; the above ii and iii steps are carried out for 40 times of circulation;
iv, stopping;
setting positive control, positive control of the test sample, negative control and blank control simultaneously in the detection process;
calculating the number N of total PCR reaction tubes, preparing N +1 parts of PCR reaction solution into a 1.5ml sterile centrifuge tube according to the reaction system, fully oscillating and uniformly mixing, centrifuging at low speed for 10s, subpackaging into PCR 8 reaction tubes, adding a positive control DNA extracting solution into a positive control tube, adding a test sample DNA extracting solution into a test sample tube, adding a test sample positive control DNA extracting solution into a test sample positive control tube, adding a negative control extracting solution into a negative control tube, adding equal volume of water into a blank control tube to replace the DNA extracting solution, and covering a reaction tube cover; fully and uniformly mixing the liquid in the PCR reaction tube, removing bubbles in the reaction tube, and centrifuging at a low speed for 30s for later use;
opening the fluorescent quantitative PCR instrument, putting the fluorescent quantitative PCR instrument into the PCR reaction tube, closing the cover, and operating the program; after the program operation is finished, analyzing an experimental result, determining a threshold line, and outputting a report of parameters including FAM fluorescence signals and Ct values;
step six, methodology control:
the result is considered invalid when one of the following conditions is not satisfied:
a. blank control: no FAM fluorescent signal is detected,
b. negative control: no FAM fluorescent signal is detected,
c. positive control: detecting FAM fluorescence signals, wherein a typical amplification curve appears in an FAM channel, the Ct value is less than or equal to 35.0,
d. positive control of test article: detecting FAM fluorescent signals, wherein a typical amplification curve appears in an FAM channel, and the Ct value is less than or equal to 35.0;
step seven, result judgment and result expression:
a. if the Ct value is less than or equal to 35.0, the tested sample is judged to be positive, namely the traditional Chinese medicine preparation is positive, and the result is expressed as 'salmonella is detected';
b. if the Ct value is more than or equal to 40.0, the sample to be detected is judged to be negative, namely the traditional Chinese medicine preparation is negative, and the result is expressed as 'no salmonella' detected;
c. if the Ct value is more than 35.0 and less than 40.0, repeating the test once, and if the Ct value is still more than 35.0 and less than 40.0 after the re-amplification, determining that the sample to be detected is the Chinese medicinal preparation is suspicious and is expressed as 'salmonella suspicious'.
5. The method of claim 4, wherein the operation of measuring the DNA concentration and purity with an ultraviolet spectrophotometer is as follows: after the ultramicro ultraviolet/visible spectrophotometer is started, entering a detection interface, adding 2ul of blank solution into a detection hole, clicking reference detection to adjust zero, and sucking liquid in the detection hole by using absorbent paper after the zero is adjusted; then adding 2ul of sample solution to be detected, clicking the sample for detection, reading and recording the obtained DNA concentration value and A280/A260 ratio, sucking the liquid in the detection hole by using absorbent paper, and repeating the operation method to continuously detect the next sample.
6. The method of claim 4, wherein:
the template DNA solution in the PCR reaction solution contains 10 ng-100 ng of DNA in each 1 mu L;
the concentration of the Taq enzyme is 5U/mu L;
the supplementing liquid is an aqueous solution containing 2 mu mol/L sodium selenite and 6mg/ml fructose;
the concentrations of the four deoxynucleotides contained in the dNTP Mix are all 10 mM;
the concentration of the detected DNA is 1.5X 10-3μg/mL~1.5×103Linear regression correlation coefficient R in mu g/mL range2Greater than 0.999;
the specificity of detecting salmonella without interference of other bacterial genes is more than 99%.
7. The method of claim 4, having a sensitivity of less than 1cfu for detecting Salmonella in a 10g sample.
8. The method according to claim 4, wherein the PCR reaction solution contains, per 25. mu.L: 0.2 mu L of template DNA solution 1 mu L, Taq enzyme, 5 mu L of 10 XPCR Buffer, 1 mu L of dNTP Mix, 1 mu L of upstream primer, 1 mu L of downstream primer, 0.5 mu L of probe, and a proper amount of 1 mu L, ddH2O aqueous solution containing 2 mu mol/L sodium selenite and 6mg/ml fructose to 25 mu L.
9. A traditional Chinese medicine pretreatment powder comprises an oral solid traditional Chinese medicine preparation, glycine and potassium chloride, and is prepared by the following steps: under the working environment and working conditions which accord with the sterility test, 10 parts by weight of oral solid traditional Chinese medicine preparation, 1.25 parts by weight of glycine and 0.5 part by weight of potassium chloride are mixed, and then are ground into uniformly mixed fine powder to obtain traditional Chinese medicine pretreatment powder; the glycine and potassium chloride were previously subjected to aseptic processing.
10. Use of the pretreatment powder of a traditional Chinese medicine according to claim 9 for the preparation of a test article for detecting salmonella contamination of a traditional Chinese medicine preparation; the method for detecting the salmonella contamination condition of the traditional Chinese medicine preparation adopts the real-time fluorescence PCR method in claim 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110078216.XA CN112391451B (en) | 2021-01-21 | 2021-01-21 | Method for pretreating Chinese medicinal preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110078216.XA CN112391451B (en) | 2021-01-21 | 2021-01-21 | Method for pretreating Chinese medicinal preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112391451A true CN112391451A (en) | 2021-02-23 |
| CN112391451B CN112391451B (en) | 2021-03-30 |
Family
ID=74624854
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110078216.XA Active CN112391451B (en) | 2021-01-21 | 2021-01-21 | Method for pretreating Chinese medicinal preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112391451B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115025231A (en) * | 2022-06-15 | 2022-09-09 | 江西省药品检验检测研究院 | Dry inoculation method of traditional Chinese medicinal materials |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705281A (en) * | 2009-11-19 | 2010-05-12 | 浙江省疾病预防控制中心 | Real-time fluorescence PCR kit used for detecting salmonella and detection method thereof |
| US20110223598A1 (en) * | 2010-03-11 | 2011-09-15 | Samsung Techwin Co., Ltd. | Method for real-time detection of salmonella in food using a cleavable chimeric probe |
| CN102676662A (en) * | 2012-04-27 | 2012-09-19 | 上海交通大学 | Method and kit for quickly detecting interior label of salmonella by adopting reverse transcription fluorescent quantitation |
| CN103233070A (en) * | 2013-04-25 | 2013-08-07 | 广州弗赛生物科技有限公司 | Specific primer, probe and kit for real-time fluorescence quantification PCR (Polymerase Chain Reaction) of salmonella |
| CN105200153A (en) * | 2015-10-29 | 2015-12-30 | 广州市疾病预防控制中心 | Gene probe for simultaneously detecting five food-borne pathogenic bacteria through liquid chip and detection method |
| CN107964566A (en) * | 2017-12-21 | 2018-04-27 | 天津宝坻紫荆科技有限公司 | Salmonella in Food PCR detection primers, probe and detection method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719424A (en) * | 2012-05-30 | 2012-10-10 | 曹际娟 | Salmonella enteritidis nucleic acid standard sample as well as building method and application thereof |
-
2021
- 2021-01-21 CN CN202110078216.XA patent/CN112391451B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705281A (en) * | 2009-11-19 | 2010-05-12 | 浙江省疾病预防控制中心 | Real-time fluorescence PCR kit used for detecting salmonella and detection method thereof |
| US20110223598A1 (en) * | 2010-03-11 | 2011-09-15 | Samsung Techwin Co., Ltd. | Method for real-time detection of salmonella in food using a cleavable chimeric probe |
| CN102676662A (en) * | 2012-04-27 | 2012-09-19 | 上海交通大学 | Method and kit for quickly detecting interior label of salmonella by adopting reverse transcription fluorescent quantitation |
| CN103233070A (en) * | 2013-04-25 | 2013-08-07 | 广州弗赛生物科技有限公司 | Specific primer, probe and kit for real-time fluorescence quantification PCR (Polymerase Chain Reaction) of salmonella |
| CN105200153A (en) * | 2015-10-29 | 2015-12-30 | 广州市疾病预防控制中心 | Gene probe for simultaneously detecting five food-borne pathogenic bacteria through liquid chip and detection method |
| CN107964566A (en) * | 2017-12-21 | 2018-04-27 | 天津宝坻紫荆科技有限公司 | Salmonella in Food PCR detection primers, probe and detection method |
Non-Patent Citations (3)
| Title |
|---|
| WONJIN KIM ET AL.: "A functional bioink and its application in myoblast alignment and differentiation", 《CHEMICAL ENGINEERING JOURNAL》 * |
| 王旭 等: "甘氨酸在氯化钾水溶液中的稀释焓", 《化学学报》 * |
| 高宣亮 等: "《食物药物毒物—关于营养和食疗的现代认识》", 30 November 1998 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115025231A (en) * | 2022-06-15 | 2022-09-09 | 江西省药品检验检测研究院 | Dry inoculation method of traditional Chinese medicinal materials |
| CN115025231B (en) * | 2022-06-15 | 2024-03-19 | 江西省药品检验检测研究院 | Dry inoculation method of traditional Chinese medicinal materials |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112391451B (en) | 2021-03-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104862393B (en) | A kind of nucleotides and its application to Cronobacter sakazakii O antigen-specifics | |
| CN101144775B (en) | Bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit | |
| CN107523619A (en) | The PCR detection kit of drug-fast bacteria comprising mcr genes and its application | |
| CN102242216B (en) | Fluorescent PCR (polymerase chain reaction) detection kit for vibrio cholerae and systematic identification method thereof | |
| CN111518877B (en) | One-tube method nest type real-time quantitative PCR detection kit for detecting echinococcus multilocularis and echinococcus granulosus by parting trace samples | |
| CN112391451B (en) | Method for pretreating Chinese medicinal preparation | |
| CN101608210B (en) | Quantitative detection kit for helicobacter pylori nucleic acid | |
| CN102676664A (en) | Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method | |
| CN109988854A (en) | For detecting oligonucleotide combinatorial, method and the kit of pathogenic Bao Te bacillus | |
| CN113584191A (en) | Primer, probe and kit for multiplex PCR detection of 7 drug-resistant genes | |
| CN110468223B (en) | Primer, probe, kit and method for detecting nucleic acid of mycoplasma pneumoniae and bauxiella based on dUTP/UNG method | |
| CN114958975A (en) | Recombinase-mediated isothermal nucleic acid amplification combination | |
| CN113512601B (en) | Molecular targets for screening for Proteus and quantitative detection methods | |
| CN112501330B (en) | Primer probe combination and kit for detecting 12 bacteria related to digestive tract infection of children | |
| CN106048051B (en) | A kind of candida krusei fluorescence PCR detection reagent kit | |
| CN108103214A (en) | The primed probe group and kit of RAA Fluorometric assay hemolytic streptococcus | |
| CN111671889A (en) | Preparation method of haemophilus parasuis type 2 inactivated vaccine | |
| CN106701976A (en) | Pathogen multiplex PCR (polymerase chain reaction) detection kit for chicken common bacterial diseases | |
| CN114107524A (en) | Characteristic sequence for rapidly detecting three pneumonia pathogenic bacteria by nucleic acid, primer and application | |
| CN116179728A (en) | Method for rapidly detecting escherichia coli in medicines by using real-time fluorescence quantitative PCR technology | |
| CN116064866B (en) | Kit, primer, method and application for detecting Burkholderia cepacia group | |
| CN113881589B (en) | Klebsiella pneumoniae KP21 strain and application thereof | |
| CN114591841B (en) | Rhodococcus equi and application thereof in preparation of inactivated vaccine | |
| CN113136444B (en) | Microdroplet digital PCR detection method for enterococcus faecalis in medical food | |
| CN201107259Y (en) | Bacteria real time fluorescent fixed quantity polyase chain reaction testing reagent box |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |