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PCR (polymerase chain reaction) method and kit for identifying spina date seeds and counterfeits thereof on basis of ITS sequence site

A technology of wild jujube seed and kit, which is applied in biochemical equipment and methods, microbiological measurement/testing, DNA/RNA fragments, etc., and can solve the problems of inconspicuous microstructure characteristics, susceptibility to interference by external factors, and identification of traits Difficult to distinguish and other problems, to achieve fast identification, reduce false positive rate, and low cost

Active Publication Date: 2016-05-25
华润三九现代中药制药有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, jujube kernel is a local customary product, and its market usage is small, but it is very similar to jujube kernel in appearance, and it is difficult to distinguish its characteristics
[0004] At present, traditional identification methods such as medicinal properties, microscopic characteristics, thin-layer chromatography, ultraviolet spectroscopy, electrophoresis, scanning electron microscopy, etc. are mostly used in the identification research of Jujube Seed. The chemical composition is complex, the specificity of the above methods is not strong, and it is easily interfered by external factors
In contrast, site-specific PCR technology is stable and not affected by environmental factors, and there are few studies on the molecular identification of jujube seed

Method used

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  • PCR (polymerase chain reaction) method and kit for identifying spina date seeds and counterfeits thereof on basis of ITS sequence site
  • PCR (polymerase chain reaction) method and kit for identifying spina date seeds and counterfeits thereof on basis of ITS sequence site
  • PCR (polymerase chain reaction) method and kit for identifying spina date seeds and counterfeits thereof on basis of ITS sequence site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, identify the primer design and synthesis of Suanzaoren

[0047] Find the ITS sequences of Suanzaoren, Lizaoren, Lentil, Hoveniae, and Bauhinia from Genbank, and construct a phylogenetic tree based on ITS, see Figure 4 , the method uses the Kimura2-parameter distance, and the confidence of each branch of the phylogenetic tree is tested by the bootstrap method, and a total of 1000 cycles are performed. It can be seen from the figure that Semanthus jujubas is closely related to Semen jujubas, followed by Hovenia dulcis, which is farther related to Zibuhinia and Lentil, and they can be distinguished with a reliability of more than 98%, which is in line with the results of plant classification. Jujube kernels, lentils, Hovenia dulcis, and Bauhinia seeds have become counterfeit products of jujube kernels. The results showed that the ITS sequence could be used to identify jujube seed and its counterfeit products.

[0048] The general primers in Table 1 were us...

Embodiment 2

[0051] Example 2, the application of primers in identification of Jujube Semen

[0052] 1. Exploration of specific primers

[0053] Extract the fruit genomic DNA of Suanzaoren, Lizaoren, Hovenia dulcis, Bauhinia and Lentil, and add them to the following reaction system for PCR amplification,

[0054] The reaction volume is 25 μL, including 12.5 μL 2x PCRSuperMix buffer (TRAN), 0.5 μL each for forward and reverse primers (0.2 μmol L -1 ), the concentration of genomic DNA was 35ng·μL -1 A total of 1.5 μL, 10 μL of sterile water.

[0055] The PCR reaction conditions were: 94°C for 5min, 35 cycles (94°C for 30s, 57°C for 15s, 72°C for 30s), 72°C for 7min.

[0056] ZmITS3 positive and negative primers for amplification results as follows figure 2 , under the above reaction conditions, only the jujube seed sample can amplify a 66bp fragment, while the counterfeit jujube kernel, Hovenia dulcis, Bauhinia seed and lentil will not amplify any bands; primer ZmITS3 can be used as the...

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Abstract

The invention discloses a PCR (polymerase chain reaction) method and kit for identifying spina date seeds and counterfeits thereof on the basis of ITS sequence site. The invention provides primers for identifying spina date seeds, particularly a primer pair capable of amplifying amplification products of nucleotide disclosed as Sequence 1 in the sequence table and nucleotide disclosed as Sequence 2 in the sequence table. The experiment proves that screening is performed on the basis of the nuclear gene ITS sequence of the original plant to obtain the site specific primers for identifying the spina date seeds and counterfeits thereof, and the PCR reaction system and conditions are regulated to establish a site-specific PCR process. The site-specific primer PCR is simple to operate, and has the advantages of low cost, favorable repetitiveness, single identification strip, low sample consumption and the like. The specific primer PCR can greatly lower the false positive rate of PCR and ensure the identification accuracy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a PCR method and a kit for identifying wild jujube kernels and counterfeit products thereof based on ITS sequence sites. Background technique [0002] Ziziphus jujuba Mill.var.spinosa (Bunge) HuexH.F.Chou is the dry mature seed of Ziziphus jujuba Mill.var.spinosa (Bunge) HuexH.F.Chou. Produced in Hebei, Shaanxi, Liaoning, Shandong, Henan, Anhui and other provinces. Suanzaoren is flat in nature, sweet in taste and sour. It has the effects of nourishing the heart, nourishing the liver, calming the mind, astringing sweat, and promoting body fluid. Modern pharmacological studies have shown that this product has the effects of sedation and hypnosis, analgesia and anticonvulsion, blood pressure reduction, blood lipid reduction, immune function enhancement, anti-hypoxia, anti-arrhythmia, anti-aging, anti-radiation and anti-platelet aggregation. [0003] Due to the reduction of wild resour...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6809C12Q1/6895
Inventor 黄璐琦袁媛张夏楠赵玉洋
Owner 华润三九现代中药制药有限公司
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