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Method for identifying commercial cigarette by applying SSR (Simple Sequence Repeat) molecular marker technique

A technology of molecular markers and technical identification, applied in the field of molecular biology, to achieve good application value, enrich the means of cigarette identification, and achieve accurate and reliable identification results

Inactive Publication Date: 2013-12-18
HONGYUN HONGHE TOBACCO (GRP) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, the authenticity of commercial cigarettes was judged based on product trademarks, packaging and smoking senses, but the use of molecular biology methods to identify cigarette raw materials has not yet been reported.

Method used

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  • Method for identifying commercial cigarette by applying SSR (Simple Sequence Repeat) molecular marker technique
  • Method for identifying commercial cigarette by applying SSR (Simple Sequence Repeat) molecular marker technique
  • Method for identifying commercial cigarette by applying SSR (Simple Sequence Repeat) molecular marker technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) SSR polymorphism primer design

[0036] Primers were designed by randomly selecting intervals with SSR sequences from the tobacco genome sequence. The whole tobacco genome sequence was downloaded from the GENBANK database, the sequence containing the SSR site was searched by the SSR Hunter software, and primers were designed on both sides of it by Oligo6.0, and the length of the target fragment was 110-250bp.

[0037] (2) Single-variety tobacco DNA extraction

[0038] Take fresh or dried single-variety tobacco leaves of Honghua Dajinyuan, NC102, K326, etc. of different years and origins, grind their tissues into fine powder, put them into a 2mL centrifuge tube, add 0.4-1mL CTAB preheated at 65°C Extraction buffer and 1-2 μL of 0.2% V / V mercaptoethanol, shake and mix well; bathe in water at 65°C for 40 minutes, mix well every 8 minutes, add an equal volume of 24:1 chloroform / isoamyl to each sample after the water bath Alcohol mixture, mix well, centrifuge at 12000r...

Embodiment 2

[0049] (1) The steps of SSR polymorphic primer design, single-variety tobacco DNA extraction, and variety-specific primer screening are the same as in Example 1.

[0050] (2) Identification of target species in mixed samples

[0051] The tobacco leaves of the three varieties were mixed in pairs in different proportions, and the mixed varieties and proportions are shown in Table 1.

[0052] Table 1 Mixed sample composition

[0053]

[0054] Extract the total DNA of the mixed sample according to the above steps, and apply the screened species-specific primer NT5 to perform PCR amplification (the steps and reaction conditions are the same as before). The obtained PCR product was subjected to capillary electrophoresis through a QIAxcel instrument, and the electrophoretic pattern was as follows: figure 2 shown.

[0055] figure 2 Among them, A01 to A03 are the mixture bands of NC102 and K326 amplified by NT5, A04 to A06 are the bands of the mixture of Honghua Dajinyuan and ...

Embodiment 3

[0057] (1) The steps of SSR polymorphic primer design, single-variety tobacco DNA extraction, and variety-specific primer screening are the same as in Example 1.

[0058] (2) Identification of target species in mixed samples

[0059] The tobacco leaves of the three varieties were mixed in pairs in different proportions, and the mixed varieties and proportions were consistent with those shown in Table 1 in Example 2.

[0060] Extract the total DNA of the mixed sample, proceed according to the above steps, and apply the screened species-specific primer NT10 for PCR amplification (the steps and reaction conditions are the same as before). The obtained PCR product was subjected to capillary electrophoresis through a QIAxcel instrument, and the electrophoretic pattern was as follows: image 3 shown.

[0061] image 3 B01 to B03 are the bands of the mixture of NC102 and K326 amplified by NT10, B04 to B06 are the bands of the mixture of Honghua Dajinyuan and K326 amplified by NT10...

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PUM

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Abstract

The invention discloses a method for identifying a commercial cigarette by applying an SSR (Simple Sequence Repeat) molecular marker technique, belonging to the field of molecular biology. The method comprises the steps of carrying out genetic map analysis on a tobacco in single variety by applying the SSR technique, screening out SSR sites with moderate aberration rate and variety specificity, extracting total DNA (deoxyribonucleic acid) of commercial cigarette shreds, amplifying by using screened specific primers, comparing genetic variation type with the type of the single variety, distinguishing the variety of tobacco leaves contained in the commercial cigarette shreds according to the variety specificity sites, and contrasting the using condition of the variety of the tobacco leaves in the corresponding cigarette formula to conveniently identify the authenticity of the cigarette. Meanwhile, the method is also suitable for the identification of tobacco leaves in single variety, tobacco shred groups and tobacco shred samples in mixed varieties.

Description

Technical field: [0001] The invention belongs to the technical field of molecular biology, in particular, the invention relates to the identification of commercial cigarettes by using a technical method of molecular biology. At the same time, the invention also relates to the identification of single-variety tobacco leaves and mixed cut tobacco samples by the method. Background technique: [0002] As one of the most important economic crops for human beings, its genetic characteristics have always been a research hotspot. At present, the complete genome sequence map of tobacco has been completed, which enables people to conduct more in-depth research on tobacco from the genome level. Among them, there are many studies on genetic diversity of tobacco varieties, but studies from the perspective of leaf group formulation and commercial tobacco have not been reported. The stability of raw material quality and the elimination of counterfeit and shoddy products are important guar...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 杨莹张天栋周博付磊王欣林赵英良汤丹瑜张玲雷声陈绍田
Owner HONGYUN HONGHE TOBACCO (GRP) CO LTD
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