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Primer and probe group and kit for detecting hemolytic streptococcus through RAA fluorescence method

A hemolytic streptococcus, primer probe technology, applied in the direction of DNA / RNA fragments, microorganisms, recombinant DNA technology, etc., can solve the problems of high false positives, complex design, etc., to achieve high specificity detection, rapid detection, large The effect of applying the foreground

Inactive Publication Date: 2018-06-01
中华人民共和国浙江出入境检验检疫局 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the design of this method is complicated, and the occurrence of false positives is high.

Method used

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  • Primer and probe group and kit for detecting hemolytic streptococcus through RAA fluorescence method
  • Primer and probe group and kit for detecting hemolytic streptococcus through RAA fluorescence method
  • Primer and probe group and kit for detecting hemolytic streptococcus through RAA fluorescence method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Select the specific conserved gene of hemolytic streptococcus as the target detection gene, and obtain the hemolytic streptococcal protease A gene through the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov). Sequence, and perform multiple sequence alignment, and select a conservative sequence, the sequence is shown in SEQ ID NO.6 in the sequence listing:

[0055] TGACAACAACAGTAGCAGCAGATGAGCTAACCACAACGAGTGAACCAACAATCACGAATCACACTCAACAACAAGCGCAACATCTCACCAATACAGAGTTGAGCTCAGCTGAATCAAAACCTCAAGACACATCACAAATCACTCTCAAGACAAATCGTGAAAAAGAGCAACCACAAGGTCTAGTCTCTGAGCCAACCACAACTGAGCTAGCTGACACAGATGCAGCACCAATGGCTAATACAGGTCCTGATGCGACTCAAAAAAGCGCTTCTTTACCGCCAGTCAATACAGATGTTCACGATTGGGTAAAAACCAAAGGAGCTTGGGACAAGGGATACAAAGGACAAGGCAAGGTTGTCG;

[0056] According to the above sequence, Sangon Bioengineering (Shanghai) Co., Ltd. was commissioned to synthesize a DNA plasmid with a size of 360bp;

[0057] According to the design principles of RAA technical primers ...

Embodiment 2

[0084] The primer probe is the same as in Example 1.

[0085] Table 4 is the composition of embodiment 2 kit

[0086]

[0087] Sample source and DNA extraction

[0088] The samples were provided by Zhejiang Entry-Exit Inspection and Quarantine Bureau. They were DNA extracted from standard strains of hemolytic streptococci and samples of hemolytic streptococci isolated from food. The extracted DNA was stored at -80°C for later use.

[0089] Take 4 reaction tubes and operate as follows respectively, absorb 27 μL of reaction buffer, add 2 μL of the mixture of probe and primer, and mix thoroughly; add 49 μL of the mixed buffer into the RAA basic fluorescent universal reaction reagent tube, Make the lyophilized powder fully dissolve and mix;

[0090] Add 1 μL negative quality control substance, 1 μL extracted hemolytic streptococcus standard strain DNA, 1 μL extracted hemolytic streptococcus sample DNA, and 1 μL positive quality control substance to the 4 prepared tubes as tem...

Embodiment 3

[0096] The sequences of the primer probe and the positive quality control product are the same as those in Example 1.

[0097] Table 5 is the composition of embodiment 3 kit

[0098]

[0099]

[0100] Sample source and DNA extraction:

[0101] The DNA of hemolytic streptococcus samples was provided by Zhejiang Entry-Exit Inspection and Quarantine Bureau, and the samples of Salmonella, Staphylococcus aureus and Escherichia coli were provided by Zhejiang International Travel Health Center. DNA was extracted by heating at 90°C for 10 minutes, and the extracted DNA was stored at -80°C spare,

[0102] Take 5 reaction tubes and operate as follows respectively, absorb 27 μL of reaction buffer, add 2 μL of the mixture of probe and primer, and mix thoroughly; add 49 μL of the mixed buffer into the RAA basic fluorescent universal reaction reagent tube, Make the lyophilized powder fully dissolve and mix;

[0103] Add 1 μL of negative quality control, 1 μL of extracted hemolytic ...

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Abstract

The invention relates to a primer and probe group and kit for detecting hemolytic streptococcus through an RAA fluorescence method, and belongs to the technical field of molecular biological detection. The primer and probe group for detecting the hemolytic streptococcus through the RAA fluorescent method comprises an upstream primer, a downstream primer and a probe; the primer and probe group is characterized in that the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1; the nucleotide sequence of the downstream primer is shown as SEQ ID NO.2; and the probe is a substance which includes the 40th-site base modified fluorescence reporter gene, the 42nd-site base modified tetrahydrofuran residue and the 43rd-site base modified quenching group, and the nucleotide sequence of the probe is shown as SEQ ID NO.3. The kit provided by the invention is rapid and sensitive in detection and simple and convenient to operate; the kit is of great significance to spread of the hemolytic streptococcus in the future; and the kit has a relatively high application prospect.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a primer probe group and a kit for detecting hemolytic streptococcus by RAA fluorescence method. Background technique [0002] Hemolytic streptococci are widely distributed in nature and exist in water, air, dust, feces, and the mouth, nose, and throat of healthy humans and animals. They can be infected through direct contact, air droplets, or through skin and mucous membrane wounds. Contaminated food such as milk, meat, eggs and their products can also infect humans. α-hemolytic streptococcus: there is a 1-2mm wide grass-green hemolytic ring around the colony, also known as type A hemolytic, and most of these streptococci are opportunistic pathogenic bacteria. β-hemolytic streptococcus: a 2-4mm wide, well-defined, completely transparent colorless hemolytic ring is formed around the colony, also known as type B hemolytic, so this kind of bacteria is also call...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12N15/11C12R1/46
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2531/119C12Q2563/107
Inventor 魏莹张小平杨永耀薛仕杰吴忠华郑伟郭利川汤赛君王智宏应清界李可谢国骏林晓霞孙丹妮
Owner 中华人民共和国浙江出入境检验检疫局
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