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One-step droplet digital PCR method for quantitatively detecting GII type norovirus in fruits and vegetables

A technology for digital and quantitative detection of droplets, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effects of good stability, good primer specificity and high reproducibility

Active Publication Date: 2017-11-10
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ddPCR is now more commonly used in the detection of genetically modified foods, but it has not been used in food-borne viruses. Therefore, if ddPCR can be applied to the detection of food-borne viruses, it will effectively expand the scope of virus detection methods. And can effectively improve the detection of viruses hidden in food inhibitors

Method used

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  • One-step droplet digital PCR method for quantitatively detecting GII type norovirus in fruits and vegetables
  • One-step droplet digital PCR method for quantitatively detecting GII type norovirus in fruits and vegetables
  • One-step droplet digital PCR method for quantitatively detecting GII type norovirus in fruits and vegetables

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 RT-ddPCR amplification annealing temperature optimization

[0056] Synthesize the primer probe according to the upstream primer and probe sequence of Norovirus GII type in Loisy and the downstream primer of Norovirus GII type in Kageyama (see Note 1, 2):

[0057] Upstream primer QNIF2: 5′-ATGTTCAGRTGGATGAGRTTCTCWGA-3′;

[0058] Downstream primer COG2R: 5′-TCGACGCCATTCTTCATTCACA-3′;

[0059] Probe QNIFs: 5′-AGCACGTGGGAGGGCGATCG-3′, the 5′ end of the probe is labeled with the fluorescent reporter group FAM, and the 3′ end is labeled with the fluorescent quencher group BHQ1. Using the same viral nucleic acid as a template, select 8 temperatures from 50 to 60°C (specifically 50°C, 51°C, 52°C, 54°C, 56°C, 57°C, 58°C, 60°C) according to the gradient temperature setting of the instrument Perform experimental optimization. The reaction conditions were: 5 μL of super mix, 2 μL of RT-Enzyme Mix, 1 μL of DTT in Bio-Rad’s One-step RT-ddPCR Advanced Kit for Probes kit, ...

Embodiment 2

[0062] Example 2 RT-ddPCR amplification primer concentration optimization

[0063] The same primer probe sequence as in Example 1 was used for the experiment, and the same viral nucleic acid was used as a template, and the primer concentrations of 0.3 μM, 0.6 μM, 0.9 μM, and 1.2 μM were selected for investigation, and the remaining reaction conditions were: Bio-Rad One-step In the RT-ddPCR Advanced Kit for Probes kit, 5 μL of super mix, 2 μL of RT-Enzyme Mix, 1 μL of DTT, the final concentration of 0.35 μmol / L probe concentration, 2 μL of template addition, supplemented with RNase Free water to 20 μL. Reverse transcription was performed at 42°C for 60 minutes, 95°C for 10 minutes to inactivate reverse transcriptase, 95°C for 30 seconds, 54°C for 1 minute, 45 cycles, 98°C for 10 minutes, and the ramp rate was 1°C / s. The results show that the reaction detection result reaches the maximum value in the 0.9 μM primer concentration, so the primer concentration of 0.9 μM ( figure 2...

Embodiment 3

[0064] Embodiment 3RT-ddPCR amplification probe concentration optimization

[0065] The same primer probe sequence as in Example 1 was used for the experiment, and the same viral nucleic acid was used as a template, and the probe concentrations of 150nM, 250nM, 350nM, and 450nM were respectively selected for investigation, and the remaining reaction conditions were: Bio-Rad One-step RT-ddPCR In the Advanced Kit for Probes kit, 5 μL of super mix, 2 μL of RT-Enzyme Mix, 1 μL of DTT, the final concentration of 0.9 μM primer concentration, 2 μL of template addition, supplemented with RNase Free water to 20 μL. Reverse transcription was performed at 42°C for 60 minutes, 95°C for 10 minutes to inactivate reverse transcriptase, 95°C for 30 seconds, 54°C for 1 minute, 45 cycles, 98°C for 10 minutes, and the ramp rate was 1°C / s. The result shows that the reaction detection result reaches the maximum in the 350nM probe concentration, so the probe concentration of 350nM ( image 3 ).

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Abstract

The invention discloses a one-step droplet digital PCR method for quantitatively detecting the GII type norovirus in fruits and vegetables. According to the method, RT-ddPCR is utilized, a primer and a probe for RT-qPCR detection of the GII type norovirus are combined for use, reaction is carried out, and then the high-sensitivity rapid detection for the GII type norovirus is realized. The method is high in sensitivity and can achieve about 2copies / [mu] L, but the sensitivity of the conventional RT-qPCR is usually 10 copies / [mu] L; the amplification efficiency is high, the amplification efficiency is 95.4%, R2 is equal to 0.9973. Under low copy number, compared with RT-qPCR, the method provided by the invention can more effectively avoid the influences of inhibitors in fruits and vegetables, and the false negative generation is reduced. Therefore, the detection method provided by the invention is sensitive, accurate and visual, a novel detection method is provided for the government departments including the agriculture department and the inspection and quarantine bureau, and related detection mechanisms and enterprises, and guiding significance is also achieved.

Description

technical field [0001] The invention relates to the technical field of food quality and safety detection, in particular to a one-step microdrop digital PCR quantitative detection method for GII type norovirus in fruits and vegetables. Background technique [0002] Foodborne viral illness is a social problem that is receiving attention. Worldwide, more than 50% of acute gastroenteritis events are caused by norovirus, the main genotype of which is GII. Norovirus was first detected by Kapikan et al. in 1972. It has the characteristics of low infectious dose and can tolerate a wide range of temperature changes. It is an important cause of non-bacterial acute gastroenteritis. Its main route of transmission is "fecal-oral", which can be transmitted directly from person to person, or through contact with contaminated water and food. Frequently contaminated foods include vegetables, soft fruits, and shellfish. Wait. How to detect and quantify norovirus in food is a hot issue. C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6851C12Q1/701C12Q2563/159
Inventor 吴希阳陈嘉茵李晖魏霜
Owner JINAN UNIVERSITY
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