Primer probe set, kit and detection method for multiple detection of human parainfluenza virus subtypes based on fluorescence RMA method

A human parainfluenza virus, multiple detection technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of complex operation procedures, inability to detect parainfluenza virus, and many false negatives

Pending Publication Date: 2021-04-09

济南国益生物科技有限公司

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AI-Extracted Technical Summary

Problems solved by technology

[0003] At present, there are many methods for detecting parainfluenza virus, but all of them have deficiencies. For example, the virus isolation and culture method takes a long time and has many false negatives, which cannot be used as a rapid diagnosis method for parainfluenza virus; the detection speed of direct immunofluorescence detection method is fast, but Sensitivity is not as good as that of tissue culture, a...

Abstract

The invention belongs to a detection method for human parainfluenza virus subtypes, and particularly relates to a primer probe group, kit and detection method for multiple detection of human parainfluenza virus subtypes based on a fluorescence RMA method. The kit comprises a detection tube containing an amplification reaction reagent, a buffer solution, magnesium acetate, a standard positive plasmid and sterile double distilled water, wherein the amplification reaction reagent comprises a primer and probe of HPIV1-3, M-MLV reverse transcriptase, escherichia coli RecA protein, UvsY protein, single-stranded binding protein GP32, Bst polymerase, exonuclease III, polyethylene oxide, trehalose, mannitol, ATP, dNTPs, creatine kinase and phosphocreatine; and the standard positive plasmid is a recombinant plasmid obtained by connecting an HN gene segment of HPIV1 with a pMD18-T vector.

Application Domain

Microbiological testing/measurementDNA/RNA fragmentation

Technology Topic

PhosphorylcreatineExonuclease III +16

Image

Examples

Experimental program(1)

Example Embodiment

[0050]Example 1

[0051]1, preparation of positive standard plasmid

[0052]Referring to the viral DNA / RNA extraction kit instruction manual, the human sub-flu virus 3 subtypes of human sub-flu virus is extracted, reflecting the reverse transcription kit specification, cDNA; CDNA is a template, PCR amplification of the HN gene, The PCR amplification product was attached to the PMD18-T vector by 1% agarose gel electrophoresis, cloning, and transformed into PMD18-T vectors, transformed into E. coli sensitized cells, blue-white spot screening, and colon colonies, colonies PCR verification. The sequencing of positive recombinant bacteria was sequencing, and the correct recombinant bacteria were cultured overnight, and plasmid DNA was extracted, and the yang-proof particles (P-1, P-2, P-3) were obtained.

[0053]2, fluorescent RMA primers and probes design

[0054]The HN-specific gene of HPIV1-3 for the human sub-flu virus is designing fluorescent RMA primers and probes, as shown in Table 2:

[0055]Table 2 primers and probes sequences

[0056]

[0057]

[0058]Note: The fluorescent group of the HPIV1-probe is modified with FAM modification, the fluorescent group of the HPIV2-probe with HEX modification, the fluorescent group of the HPIV3-probe with ROX modification, the quenching group is modified with BHQ, 3 'end Blocked the group C3-Spacer modification.

[0059]3. Establishment of fluorescence RMA reaction system

[0060]42.5 μL of buffer and 5 μl of extracted viral RNA template were added to a constant temperature amplification reaction tube containing a primer probe enzyme, mixed; at the end of the tube, 2.5 μl of 280 mM magnesium acetate solution was added and mixed; The above reaction tubes were placed in a fluorescent detector and reacted at 42 ° C for 20 min; each reaction was used as a positive control with a standard yang-based grain, and sterile double vapor is a negative control.

[0061]4, judgment

[0062]According to whether or not the corresponding amplification curve occurs, it is analyzed whether or not a sub-flu virus is contained in the sample; only the HPIV1 subtype amplification curve is characterized by HPIV1 positive; only HPIV2 subtype amplification curves appear. It is characterized by HPIV2 positive; only HPIV3 subtype amplification curves appear as HPIV3 positive.

[0063]5, fluorescence RMA method to detect the sensitivity analysis of HPIV1-3

[0064]10 times series dilution of the standard yang nature (including 10)4103102101And 100Copy / Reaction), with sterile double vapor as a negative control, a fluorescent RMA reaction was performed under the above reaction system conditions, each concentration repeated 3 tests. according toFigure 1-3 It can be seen, 104-101The results were positive, i.e., the sensitivity of the fluorescent RMA test tester reached 10 copies / reactions.

[0065]6. Specific analysis of HPIV1-3 by fluorescence RMA method

[0066]HPIV1, HPIV2, HPIV3, HPIV3, HPIV3, HPIV3, A-influenza virus (Flua), Brea virus (FLUB), respiratory nucleic acid samples, and evaluation methods are detected, respectively. The bacteria double vapor is a negative control, each test repeatedly detected 3 times. according toFigure 4-6It can be seen that only when the target pathogen is HPIV1, the fluorescent RMA method is detected as HPIV1 positive, and the HPIV2, HPIV3 and other viruses are negative; only when the target pathogen is HPIV2, it is detected as HPIV2 positive, and HPIV1, HPIV3 and other Virus detection is negative; when the target pathogen is HPIV3, it is detected as HPIV3 positive, and HPIV1, HPIV2 and other viruses were negative. Note The fluorescent RMA method has good detection effects and specificity.

PUM

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