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Human astrovirus type 1 nucleic acid detection standard substance and preparation method and application thereof

An astrovirus and detection standard technology, applied in the field of human astrovirus type 1 nucleic acid detection standard substance and its preparation, to achieve the effects of avoiding biological safety, easy preparation, and good uniformity

Pending Publication Date: 2018-05-29
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no standard material using human astrovirus type 1 RNA fragments as raw materials at home and abroad

Method used

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  • Human astrovirus type 1 nucleic acid detection standard substance and preparation method and application thereof
  • Human astrovirus type 1 nucleic acid detection standard substance and preparation method and application thereof
  • Human astrovirus type 1 nucleic acid detection standard substance and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 is used for developing the specific primer and probe of astrovirus type 1 nucleic acid detection standard substance

[0044] The target sequence encoding caspidprotein of human astrovirus type 1 open reading frame 2 (Open Reading Frame, ORF2) was selected, and sequence analysis and alignment were carried out through NCBI online tools, using Prime Express software V4.0 (ABI, Foster City, CA, USA) designed more than 10 pairs of primers and probe combinations, and after screening, finally obtained a highly specific standard substance for the preparation of human astrovirus type 1 nucleic acid detection standard substances and subsequent reverse transcription droplet digital polymerase chain reaction (Reverse Transcript-droplet digital Polymerase Chain Reaction, RT-ddPCR (RT-ddPCR) detection method with 1 set of primers and probe combinations, the sequence is shown in Table 1. The 5' end of the probe is labeled with FAM, and the 3' end is labeled with BHQ. Pri...

Embodiment 2

[0047] Example 2 Preparation of Human Astrovirus Type 1 Nucleic Acid Detection Standard Substance Candidates

[0048] Extract viral RNA from the biological sample (mostly stool sample) that is identified as human astrovirus type 1 positive, utilize the specific primer amplification shown in SEQ ID No.2 and SEQ ID No.3 that screens out as SEQ ID No. The DNA fragment shown in ID No.5; then connect the DNA fragment with a plasmid vector to construct a recombinant plasmid, and transform the recombinant plasmid into Escherichia coli competent cells to construct Escherichia coli containing the recombinant plasmid; through the propagation of Escherichia coli , which can realize the large-scale preparation of recombinant plasmids of the above DNA fragments; extract the recombinant plasmids, and then use HamHI restriction endonuclease to single-enzyme digest the recombinant plasmids to form in vitro transcription templates, and obtain human astroviruses through in vitro transcription an...

Embodiment 3

[0087] The establishment of embodiment 3RT-ddPCR method

[0088] The RT-ddPCR method is mainly used for the study of uniformity, stability and value determination in the preparation process of standard substances for human astrovirus nucleic acid detection. The optimal detection range of RT-ddPCR is 20-2000 copies / μL. Use the balance weighing method to carry out gradient dilution of the RNA standard substance. Refer to the method of the One-Step RT-ddPCR Kit for Probes kit to prepare the standard substance above. For RT-ddPCR detection, the primers and probes used are the same as shown in Table 1. Refer to the kit method. The specific operation method is as follows:

[0089] 1. PCR reaction system

[0090] 2×one-step RT-ddPCR supermix 10μL, 25mM manganese acetate solution0.8μL, forward primer and reverse primer (as shown in SEQ ID No.2 and SEQ ID No.3) each 1.0-1.8μL, fluorescent probe (As shown in SEQ ID No.4) 0.5 μL, RNA template 2.0 μL, DEPC water to make up to 20 μL. Th...

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Abstract

The invention discloses a human astrovirus type 1 nucleic acid detection standard substance, a preparation method thereof, and application of the human astrovirus type 1 nucleic acid detection standard substance serving as the standard substance in human astrovirus type 1 nucleic acid detection. The RNA sequence of the human astrovirus type 1 nucleic acid detection standard substance is as shown in SEQ ID No. 1. The human astrovirus type 1 nucleic acid detection standard substance is free of organism infectivity, easy to prepare, good in uniformity, sufficient in stability, accurate, traceable, capable of being used as the positive control of human astrovirus type 1 nucleic acid detection and the external standard substance of the human astrovirus type 1 nucleic acid detection, capable ofbeing used for evaluating new human astrovirus type 1 nucleic acid detection methods, capable of providing a substantial foundation for the certification and accreditation of human astrovirus type 1 nucleic acid detection ability and widely applicable to various laboratories.

Description

technical field [0001] The invention relates to the technical field of biological detection. More specifically, it relates to a human astrovirus type 1 nucleic acid detection standard substance and its preparation method and application. Background technique [0002] Appleton et al. first discovered Human Astrovirus (HAstV) in the feces of patients with gastroenteritis in 1975, and there are outbreaks of Astrovirus infection to varying degrees in various countries every year. Astrovirus is a single-stranded positive-strand RNA virus without a capsid, with a diameter of 28-35nm and 5-6 small horns, making the entire virus particle a star-shaped structure. The viral gene is 6.8kb in length, including a 5' non-coding region (NCR), three open reading frames (ORFs) ORF1a, ORF1b and ORF2, and 80 nucleotides of 3 'noncoding regions and a polyA tail of approximately 30 nucleotides). Astroviruses can be divided into eight serotypes, HAstV-1 to HAstV-8. At present, Human Astroviru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/166
Inventor 徐蕾蕊曾静汪琦付溥博
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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