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A kit for the quantitative detection of sweet potato components based on microdroplet digital PCR and its application

A quantitative detection and micro-droplet digital technology, which is applied in the field of molecular biology detection, can solve the problems of unsatisfactory detection effect and achieve good specificity, high detection sensitivity, and simple operation steps

Active Publication Date: 2022-03-29
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are two problems in the current market: on the one hand, other sources of low-value starch such as cassava and corn starch are mixed in the sweet potato processing process; on the other hand, starches with relatively high cost such as horseshoe starch are mixed sweet potato starch
However, when the above technique is used for the quantitative detection of sweet potato source components in mixed samples, the detection effect is not ideal

Method used

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  • A kit for the quantitative detection of sweet potato components based on microdroplet digital PCR and its application
  • A kit for the quantitative detection of sweet potato components based on microdroplet digital PCR and its application
  • A kit for the quantitative detection of sweet potato components based on microdroplet digital PCR and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 specificity test

[0059] Genomic DNA was extracted from sweet potato, purple potato, cassava, potato, yam, taro, pea, sorghum, carrot, tomato, rice, soybean and barley crops, and the upstream primer (attaatgcac ttttaaaggcggac, SEQ ID No.1), the downstream primer of β-AMY gene (ccctcccaat accgtccat, SEQ ID No.2) and the probe of β-AMY gene (tgggtacaca gtagaaggat tgtcattttc c, SEQ ID No.3) carry out fluorescent quantitative PCR amplification . The reaction program of the fluorescent quantitative PCR is as follows: 95°C, 5min, 1°C / s; 94°C, 15s, 1°C / s, 60°C, 1min, 1°C / s, a total of 40 cycles; 98°C, 10min, 1°C / s.

[0060] Among the above-mentioned 13 crops, only sweet potato and purple potato samples were specifically detected (sweet potato and purple potato with typical S-shaped amplification curves), and purple potato is a kind of sweet potato, and no other crops were detected (see figure 1 ). This shows that the primer probe designed in the present inven...

Embodiment 2

[0061] The linear relationship between embodiment 2 sweet potato quality (mg) and copy number concentration (copies / μL)

[0062] Weigh 5mg, 15mg, 30mg, 40mg, 50mg of gradient quality sweet potato powder samples, use Wizard Genomic DNA purification kit (Promega, A1120) to extract genomic DNA, use 20μL digital PCR reaction system (2×ddPCRTM master mix 10μL; concentration 10μmol / μL 0.8 μL of each primer, 0.4 μL of probe with a concentration of 10 μmol / μL, 2 μL of DNA template, and replenish water to 20 μL. The ddPCR reaction conditions are: 95°C, 5min (1°C / s); 94°C, 15s (1°C / s ), 60°C, 1min (1°C / s), a total of 40 cycles; 98°C, 10min (1°C / s), 12°C to store the reaction product.)

[0063] The obtained data are shown in Table 1, and the linear relationship data between the sweet potato mass (mg) and the copy number concentration (copy number / μL) are established as figure 2 . The linear relationship between the quality of sweet potato and the copy concentration of β-AMY gene is y=...

Embodiment 3

[0066] The LOD and LOQ determination of the sweet potato DNA concentration detection of embodiment 3

[0067] The DNA concentration gradients were 34, 6.8, 1.36, 0.27 and 0.09ng / μL, and each gradient was set up in three parallels. The data are shown in Table 2 and image 3 , it can be seen from Table 3 that the LOQ can be detected in 10 parallel experiments and the RSD of the copy number concentration of the parallel experiments is less than 25%, which is 2.77 copies / μL.

[0068] LOD is the lowest copy number concentration that can be detected in no less than 9 out of 10 parallels. The test results of sweet potato DNA concentration gradient LOD and LOQ are shown in Table 3, Figure 4 and Figure 5 . It can be seen from Table 3 that the LOD is that the lowest copy number concentration that can be detected in 10 parallels is 0.81 copy / μL.

[0069] Table 2 Test results of sweet potato DNA concentration and copy number

[0070]

[0071]

[0072] Table 3 Test results of ...

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Abstract

The invention provides a kit for quantitatively detecting sweet potato components based on droplet digital PCR and an application thereof, belonging to the field of molecular biology detection. The test kit for the quantitative detection of sweet potato components based on droplet digital PCR, including the upstream primer of the BETA-AMY gene, the downstream primer of the β-AMY gene and the probe of the β-AMY gene; application. The droplet digital PCR method for the quantitative detection of sweet potato components uses a digital PCR system to detect the fluorescent signal of the sweet potato-specific single-copy species gene β-AMY, and the measured copy number concentration of the sweet potato-specific single-copy species gene β-AMY, according to the copy number concentration (copy number / μL) and sweet potato mass (mg) to calculate the content of sweet potato components. The invention can carry out specific quantitative detection on sweet potato components, and has the characteristics of simplicity, speed and high detection sensitivity.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a kit for quantitatively detecting sweet potato components based on droplet digital PCR and an application thereof. Background technique [0002] Sweet potatoes are rich in starch, and sweet potato starch is one of the most nutritious foods in crops. However, there are two problems in the current market: on the one hand, other sources of low-value starch such as cassava and corn starch are mixed in the sweet potato processing process; on the other hand, starches with relatively high cost such as horseshoe starch are mixed Into the lower price of sweet potato starch. [0003] In the prior art, methods for determining sweet potato source components include ordinary biological microscopy, scanning electron microscopy, laser particle size analysis, rapid viscosity analysis, small-angle X-ray scattering (SAXS), and real-time fluorescent PCR techniques. However, when the ab...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6851
CPCC12Q1/6895C12Q1/6851C12Q2531/113C12Q2537/16C12Q2563/159
Inventor 刘二龙李志勇吕英姿董旭婉刘婧文关丽军
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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