Probe primer as well as fluorescent quantitative PCR (polymerase chain reaction) detection method and application thereof for identifying bulbus fritillariae cirrhosae primitives

Abstract

The invention provides a probe primer as well as a fluorescent quantitative PCR (polymerase chain reaction) detection method and application thereof for identifying bulbus fritillariae cirrhosae primitives. The probe primer is composed of DNA (deoxyribonucleic acid) sequences as shown in SEQ ID NO: 1-21. The detection method is strong in specificity and high in sensitivity, can be used for qualitative identification of different basic sources of the bulbus fritillariae cirrhosae medicinal material, and has relatively strong innovativeness and application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to a drug identification and detection method, in particular to a probe primer and a fluorescent quantitative PCR detection method and application thereof for identifying different origins of Fritillaria cirrhosa. BACKGROUND

[0002] Fritillaria cirrhosa is a commonly used medicinal material and is regarded as a good medicine for relieving cough and reducing sputum. The origin of Fritillaria cirrhosa recorded in Chinese Pharmacopoeia includes six species of plants in the Liliaceae family, i.e., Fritillaria cirrhosa D.Don, Fritillaria unibracteata Hsiao et K.C.Hsia, Fritillaria przewalskii Maxim., Fritillaria delavayi Franch., Fritillaria taipaiensis P.Y.Li or Fritillaria unibracteata Hsiao et K.C.Hsiavar.wabuensis (S.Y.Tang et S.C.Yue) Z.D.Liu, S.Wang et S.C.Chen, and can be divided into Songbei, Qingbei, Lubei and cultivated products according to the differences in properties. It is traditionally believed that Songbei and Qingbei are the best, and the market price of Songbei and Qingbei is the highest, and the phenomenon of other Fritillaria cirrhosa being imitated or adulterated is relatively common. Songbei and Qingbei correspond to Fritillaria cirrhosa (origin name), Fritillaria unibracteata and Fritillaria przewalskii. SUMMARY

[0003] The application provides a probe primer and a fluorescent quantitative PCR detection method and application thereof for identifying different origins of Fritillaria cirrhosa, thereby providing technical support for quality evaluation and grade division of Fritillaria cirrhosa, and having important practical value and application prospect.

[0004] In one aspect, the application provides a probe primer. The probe primer is composed of a DNA sequence shown in SEQ ID NO: 10-21.

[0005] In another aspect, the application provides a fluorescent quantitative PCR detection method for identifying different origins of Fritillaria cirrhosa. The method comprises the following steps:

[0006] weighing Fritillaria cirrhosa samples and sample pretreatment;

[0007] extracting DNA from the samples and preparing a DNA template; and

[0008] selecting a probe primer;

[0009] optimizing reaction conditions;

[0010] According to the case of the amplification curve, it is determined whether the Fritillariae cirrhosae medicinal material sample source is Fritillariae cirrhosae / Fritillariae walujinii / Fritillariae przewalskii.

[0011] The method can be used for detecting whether the sample is the three original sources of Fritillariae cirrhosae; and the probe primer is a probe primer with a sequence number of SEQ ID NO 10-21.

[0012] In some embodiments, each set of probe primers includes three sequences, and when any one set is used for detection, the three sequences included therein are used simultaneously without splitting.

[0013] In some embodiments, the sample is a traditional Chinese medicinal material.

[0014] In some embodiments, the step of extracting DNA uses ultraviolet spectrophotometry for quantitative and purity determination.

[0015] In some embodiments, the reaction conditions include: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 60℃ annealing for 30-60s, and 60℃ fluorescence collection. According to the nature of the selected reaction premix, the reaction conditions can be adjusted.

[0016] In some embodiments, each set of probe primers includes three sequences, and when any one set is used for detection, the three sequences included therein are used simultaneously without splitting.

[0017] In some embodiments, the sample to be detected is a traditional Chinese medicinal material.

[0018] In some embodiments, the sample to be detected is a traditional Chinese medicinal material.

[0019] In some embodiments, the sample to be detected is a traditional Chinese medicinal material. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The detection effect of the probe primers on the six different original sources of Fritillariae cirrhosae and other Fritillariae.

[0021] Figure 2 The method applicability test. The Fritillariae cirrhosae, Fritillariae walujinii and Fritillariae przewalskii from different producing areas and sources were tested, and each sample appeared positive amplification;

[0022] Figure 3For the method detection limit test, the DNA concentrations were 100 ng, 10 ng, 1 ng, 0.1 ng and 0.01 ng from 1 to 5, respectively; the DNA concentration of 0.01 ng could still be detected, indicating that the method had high sensitivity;

[0023] Figure 4 For the detection limit test of the common probe primer of Fritillaria, the DNA concentrations were 100 ng, 10 ng, 1 ng, 0.1 ng and 0.01 ng from 1 to 5, respectively; the DNA concentration of 0.01 ng could still be detected, indicating that the probe primer had high sensitivity;

[0024] Figure 5 For the specific probe primer and the common probe primer, the amplification efficiency was compared.

[0025] Figure 6 For the specific probe primer and the common probe primer, the amplification efficiency was compared. DETAILED DESCRIPTION

[0026] The application will be further described in detail below in combination with the drawings and examples, and these examples are merely illustrative, and therefore should not be interpreted as limiting the scope of the application.

[0027] I. Instruments and reagents

[0028] Real-time fluorescence quantitative PCR instrument (AB 7500 FAST), analytical balance (Mettler AB135-S), pure water instrument (Millipore Corporation), ball mill (M400, Retsch), micro ultraviolet spectrophotometer, high-speed centrifuge, metal heater, plant genome extraction kit (DP321, Tiangen Biochemical Technology Co., Ltd.), Universal Master Mix II.

[0029] II. Sample collection

[0030] More than 100 batches of samples were collected, including 6 provenance samples of accurate source of Fritillaria cirrhosa, Fritillaria walujewii, Fritillaria pallidiflora and other common medicinal Fritillaria, and commercial samples. See the sample information table in annex 1 for specific information.

[0031] III. Sample processing and DNA extraction

[0032] 1. Sample processing and DNA extraction

[0033] The test sample was ground into fine powder, and 20 mg of the fine powder was precisely weighed and used to extract DNA using a plant genomic extraction kit to obtain a test sample solution. The Fritillaria cirrhosa control medicinal material was ground into fine powder, and 20 mg of the fine powder was precisely weighed and used to prepare a positive control solution in the same manner. If it is a prepared formula, the sampling amount can be appropriately increased to 50-200 mg.

[0034] 2. Specific steps for DNA extraction:

[0035] 400 μl of buffer FP1 and 6 μl of RNase A (10 mg / ml) were added, vortexed for 1 min, and left at room temperature for 10 min. 130 μl of buffer FP2 was added, mixed well, and vortexed for 1 min. Centrifugation was performed at 12,000 rpm (~ 13,400 x g) for 5 min, and the supernatant was transferred to a new centrifuge tube; the above steps were repeated. 0.7 times the volume of isopropanol was added to the supernatant (for example, 500 μl of supernatant plus 350 μl of isopropanol), mixed well by inverting, centrifuged at 12,000 rpm (~ 13,400 x g) for 2 min, and the supernatant was discarded, and the precipitate was retained. 600 μl of 70% ethanol was added, vortexed for 5 sec, centrifuged at 12,000 rpm (~ 13,400 x g) for 2 min, and the supernatant was discarded; the above steps were repeated. The cap was opened and inverted, and the residual ethanol was completely dried at room temperature for 5-10 min. 100 μl of elution buffer TE was added, and the DNA was dissolved in a water bath at 65°C for 10-30 min to obtain a DNA solution, which was stored at 4°C or -20°C.

[0036] Silica gel centrifugal column method can also be used for extraction. For prepared formulas, optimized CTAB extraction methods and the like can be used to obtain a DNA template solution with sufficient purity (generally, OD260 / 280 is required to be between 1.7 and 1.9).

[0037] Four, establishment of a fluorescent quantitative PCR detection method

[0038] 1. Probe primer design and screening

[0039] According to the DNA sequence analysis and comparison of the ITS and psbA-trnH regions, probe primers were designed in the intraspecific conserved and interspecific different regions, wherein the 5' end of the probe was labeled with a fluorescein group FAM or VIC, and the 3' end was labeled with a non-fluorescein quenching group BHQ1. Two or more sets of probe primers were designed for each target species.

[0040] The probe primer sequence information of the present application is shown in Annex 2.

[0041] 2. PCR reaction system and reaction conditions

[0042] The reaction system, with a volume of 20 μL, includes 10 μL of fluorescent PCR reaction mix (2×), 0.5 μL each of upstream and downstream primers and probe (10 μmol / L), 1 μL of template DNA, and autoclaved ultrapure water to a total volume of 8.1 μL. The reaction solutions for the test samples and positive controls were prepared according to the above system. The blank control consisted of a reaction system without template DNA, in which an equal volume of autoclaved ultrapure water was used instead of template DNA. Three replicates were performed for each sample and control, and the CT value was the average of the three replicates.

[0043] Reaction conditions: 50°C for 2 minutes; 95°C for 10 minutes; 95°C for 15 seconds, 60°C for 1 minute, 40 cycles.

[0044] Table 1. Applicability of Fritillaria universal probe primers

[0045]

[0046] V. Methodological Investigation

[0047] 1. Investigation of identification effect

[0048] Using this method, the three origins of Songbei / Qingbei (Chuan Fritillaria, Dark Purple Fritillaria, Gansu Fritillaria) showed typical amplification curves, while the amplification curve of Wabu Fritillaria was relatively low and clearly separated from the above three species. No obvious positive amplification curves were found for other Fritillaria ( Figure 1 ).

[0049] 2. Applicability assessment

[0050] The specific probe primers for Fritillaria cirrhosa were used to detect Fritillaria cirrhosa, Fritillaria thunbergii and Fritillaria dasyphylla from different origins and sources, and each sample showed obvious positive amplification ( Figure 2 The Fritillaria universal probe primers were used to detect various medicinal materials from the genus Fritillaria, and obvious positive amplification was observed for each medicinal material (Table 1).

[0051] 3. Detection limit investigation

[0052] The detection limit of the specific probe primer of Fritillaria cirrhosa was tested, and the DNA concentrations from 1 to 5 were 100ng, 10ng, 1ng, 0.1ng, and 0.01ng respectively; the DNA concentration was still detectable at 0.01ng ( Figure 3 ).

[0053] The detection limit of Fritillaria universal probe primer was tested, and the DNA concentrations from 1 to 5 were 100ng, 10ng, 1ng, 0.1ng, and 0.01ng respectively; the DNA concentration was still detectable at 0.01ng ( Figure 4 ).

[0054] Fritillariae cirrhosae and common adulterant Fritillariae ussuriensis were mixed in different proportions, and specific probe primer (2) and general probe primer (1) were used for detection. The results showed that the specific probe primer (2) could still detect Fritillariae cirrhosae when the proportion was 0.1%, Figure 5

[0055] 4. Amplification efficiency

[0056] The amplification efficiency of specific probe primer and general probe primer in the same reaction system was compared. The results showed that the amplification efficiency of the two was 92.2% and 96.6, respectively, indicating that the efficiency of the two was close and could be used for quantitative detection (Table 2, Figure 6

[0057] Table 2. Comparison of amplification efficiency of specific primer and general primer

[0058]

[0059] 5. Determination method

[0060] For qualitative detection, the CT value critical point can be set at 35, and CT < 35 is considered to detect Fritillariae cirrhosae and Fritillariae ussuriensis.

[0061] Table 3. Absolute value of △CT corresponding to different adulteration proportions

[0062]

[0063] Appendix:

[0064] Appendix 1. Information table of medicinal material samples

[0065]

[0066]

[0067]

[0068]

[0069] Appendix 2. Probe primer sequence information

[0070]

[0071]

[0072] Some specific embodiments are described in detail in this paper, however, this is only as an example to illustrate the purpose of the invention, and does not limit the scope of the following claims. It should be understood that different substitutions, changes and modifications of the specific schemes described herein do not deviate from the connotation and extension defined by the claims of the invention, and therefore should belong to the scope of the invention claimed in this application.​​

Claims

1. A probe primer, characterized in that The probe primers consist of the DNA sequences shown in SEQ ID NOs: 1-21.

2. A fluorescence quantitative PCR detection method for distinguishing between Fritillaria cirrhosae origins, characterized in that: The steps include: Weighing Fritillaria samples and sample pretreatment; extracting DNA from the sample and preparing a DNA template; and Select probe primers; Optimize reaction conditions; Different judgment criteria are set according to different detection requirements. Different judgment criteria are set according to different testing requirements. For qualitative testing, the CT value is used to determine whether it is dark purple Fritillaria / Gansu Fritillaria. Wherein, the method can be used to identify different origins of Fritillaria cirrhosa; the probe primer is the probe primer according to claim 1.

3. The method according to claim 2, wherein each set of probe primers comprises three sequences, and when any one set is used for detection, the three sequences comprised therein are used simultaneously without being separated. The method according to claim 2 , wherein the sample is a Chinese medicinal material.

5. The method according to claim 2, wherein the step of extracting DNA is performed by ultraviolet spectrophotometry for quantitative analysis and purity determination.

6. The method according to claim 2, wherein the reaction conditions include: Pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C for 30-60 seconds, and fluorescence collection at 60°C. Reaction conditions can be adjusted depending on the properties of the selected reaction premix.

7. A use of a probe primer, characterized in that, The probe primer is the probe primer set according to claim 1; the probe primer is used to identify the origin of Fritillaria cirrhosa through a fluorescent quantitative PCR detection method.

8. The method according to claim 7, wherein each set of probe primers comprises three sequences, and when any one set is used for detection, the three sequences comprised therein are used simultaneously without being separated.

9. The method according to claim 7, wherein the sample to be tested is a Chinese medicinal material.

10. A kit, characterized in that The kit contains the probe primer according to claim 1 and is used for distinguishing between Fritillaria cirrhosae origins.

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