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Primer, probe and kit for detecting rotavirus and Norovirus liquid phase chips

A rotavirus and chip detection technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve difficult problems and achieve low detection costs, strong specificity, and high sample efficiency. less effect

Inactive Publication Date: 2011-08-17
何雅青 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with conventional PCR, fluorescent quantitative PCR has advantages in sensitivity, specificity, and speed. However, real-time fluorescent PCR technology is limited by the type of fluorescence and the instrument itself. It can only detect up to 5 targets, and the success of the experiment is extremely difficult. Big

Method used

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  • Primer, probe and kit for detecting rotavirus and Norovirus liquid phase chips
  • Primer, probe and kit for detecting rotavirus and Norovirus liquid phase chips
  • Primer, probe and kit for detecting rotavirus and Norovirus liquid phase chips

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: the construction of type A rotavirus, GI type norovirus and GII type norovirus positive standard: respectively use SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO 8. The amplified product obtained by the primer pair of sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 is the recombinant plasmid constructed by the insert as a positive standard, specifically as follows.

[0023] 1) Primer sequence:

[0024] KHRV-A F 5'-CATGTTGATTAAGTGAGGACCAGAC-3'; namely SEQ ID NO:1

[0025] KHRV-A R 5'-CAGTTACTCTACGTAGCGAACATG-3'; ie SEQ ID NO:2

[0026] KNoV GI F 5'-AGGGTGGCAGGCCATGTT-3'; namely SEQ ID NO:7

[0027] KNoV GI R 5'-TCACCGGGGGTATTATTTGG-3'; ie SEQ ID NO:8

[0028] KNoV GII F 5'-CCTGCACCTCCCAATGGAA -3'; namely SEQ ID NO:9

[0029] KNoV GII R 5'-AATCCAGGGGTCAAATTACATTTTG-3'; namely SEQ ID NO: 10.

[0030] 2) Extract rotavirus, norovirus type GI, and norovirus GII RNA respectively, reverse transcribe into cDNA, and perform PCR amplification with corres...

Embodiment 2

[0039] Example 2: Establishment of multiplex PCR amplification and liquid-phase chip detection method for rotavirus and norovirus.

[0040] 1) The base sequences of the primers that specifically amplify the sequences of type A rotavirus, type GI norovirus and type GII norovirus are:

[0041] HRV-A F 5'-CATGTTGATTAAGTGAGGACCAGAC-3'; namely SEQ ID NO: 1

[0042] HRV-A R 5'-CAGTTACTCTACGTAGCGAACATG-3'; namely SEQ ID NO:2

[0043] NoV GI F 5'-GCTGGATGCGCTTCCATGACC-3'; namely SEQ ID NO:3

[0044] NoV GI R 5'-TCCGGTACCARCTGRCCRGC-3'; namely SEQ ID NO:4

[0045] NoV GII F 5'-GCCAATGTTCAGATGGATGAGAT-3'; namely SEQ ID NO:5

[0046] NoV GII R 5'-TCTTCATTCACAAAAYTGGGAG-3; namely SEQ ID NO:6

[0047] The 5' ends of the three downstream primers were labeled with Biotin.

[0048] 2) Multiplex PCR amplification.

[0049] Preparation of mixed primer working solution: the primer ratio is upstream primer: downstream primer = 1μM: 10μM = 1:10 for amplification; mix rotavirus, GI type norov...

Embodiment 3

[0085] Example 3: Sensitivity experiment for detection of rotavirus and norovirus liquid-phase chip.

[0086] The positive standard substance prepared in Example 1 is quantified, and diluted by 10-fold dilution method, and diluted to 10 -5, namely 100ng / 5μL. Carry out multiplex PCR amplification and liquid phase chip detection with the detection method of embodiment 2, the amount of template added to each PCR reaction is 5 μ L, PCR product and the microsphere mixture of coupling specific probe carry out hybridization reaction, then use liquid phase chip detection instrument to test. The sensitivity test results of liquid chip detection for rotavirus, GI type norovirus and GII type norovirus are shown in Table 5-7 respectively.

[0087] Table 5 Sensitivity experiment results of rotavirus liquid-phase chip detection.

[0088] Sample

[0089] Table 6 Sensitivity test results of GI type norovirus liquid chip detection.

[0090] Sample

[0091] Table 7 Sensi...

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Abstract

The invention provides a primer, a probe and a kit for detecting rotavirus and Norovirus liquid phase chips. The primer comprises three pairs of primers of a specific amplification A type rotavirus sequence, a GI type Norovirus sequence and a GII type Norovirus sequence. The probe comprises three specificity detection probes of A type rotavirus, GI type Norovirus and GII type Norovirus. The kit comprises the three pair of primers and a specificity detection microsphere mixture prepared by coupling the three specificity detection probes and a fluorescence coding microsphere. The experiment proves that the primer and the probe disclosed by the invention are characterized in that a method of combining multiple PCR (polymerase chain reaction) with liquid phase chip detection can simultaneously and accurately detect the A type rotavirus, the GI type Norovirus and the GII type Norovirus. The primer, the probe and the kit have the advantages of strong specificity and high sensitivity.

Description

technical field [0001] The invention relates to biological detection technology, in particular to primers, probes and kits for liquid phase chip detection of rotavirus and norovirus. Background technique [0002] Acute diarrhea is a serious global public health problem, and its incidence in children ranks second only to respiratory tract infection. Children under 5 years of age are particularly prominent in developing countries, with an annual incidence rate of 2.2. There are 3-5 billion cases every year in the world, of which 5-10 million die. The pathogens include bacteria, viruses and protozoa, etc., and viruses are the most serious. Group A rotaviruses are the leading cause of severe gastroenteritis (diarrhea) in children, accounting for 20-60% of hospitalized children with diarrhea. [0003] Rotavirus (rotavirus, RV) is a virus belonging to the genus Rotavirus in the Reoviridae family. The virion is spherical and has a double-layer capsid, and each layer of capsid is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 何雅青胡春凌汪朝晖
Owner 何雅青
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