Human cytomegalovirus nucleic acid quantitative detection primer and probe, kit and use thereof

A technology of human cytomegalovirus and detection kit, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., and can solve problems such as false positives and cross-contamination

Active Publication Date: 2016-01-20
厦门安普利生物工程有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the amplified product needs to be detected by electrophoresis, it is easy to cause cross-contamination and false positive results

Method used

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  • Human cytomegalovirus nucleic acid quantitative detection primer and probe, kit and use thereof
  • Human cytomegalovirus nucleic acid quantitative detection primer and probe, kit and use thereof
  • Human cytomegalovirus nucleic acid quantitative detection primer and probe, kit and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Preparation of the kit

[0080] Composition of various reagents:

[0081] Lysis solution: (10mMPH7.0), silica (1 weight‰), TritonX-100 (1vol‰), EDTA·2Na (10mM), guanidine isothiocyanate (2M);

[0082] detergent: (10mMPH7.0) (sigma), TritonX-100 (1 volume‰), EDTA·2Na (1mM), guanidine isothiocyanate (2M);

[0083] Repeat washing liquid: (10mMPH7.0), TritonX-100 (1 volume‰), EDTA·2Na (1mM)

[0084] HCMVPCR reaction tube: Taq enzyme (2U), UNG enzyme (0.01U), EDTA·2Na (0.1mM), paraffin oil (25 microliters);

[0085] HCMV reaction buffer: HCMV upstream and downstream primers (10pM), HCMV probe (5pM), (20mMPH9.0), KCl (50mM), MgCl2 (1.5mM), EDTA·2Na (0.1mM), dNtps (0.2mM);

[0086] HCMV quantitative standards are shown in Table 2

[0087]

[0088] HCMV negative control: normal saline;

[0089] HCMV critical positive control: normal saline, 5.0×10 3 copies / mL of HCMV plasmid;

[0090] HCMV strong positive control: normal saline, 5.0×10 7 HCMV plasmid in copies / mL.

[0091] The p...

Embodiment 2

[0098] Example 2: Application of the kit for sample detection

[0099] (1) Sample

[0100] 1. 8 samples to be tested are negative (Xiamen City Hospital of Traditional Chinese Medicine) Clinical collection of HPV6 vaginal secretion samples, HPV11 vaginal secretions samples, HPV16 vaginal secretions samples, HPV18 vaginal secretions samples, Chlamydia trachomatis secretions samples, Secretion samples of Neisseria gonorrhoeae, Ureaplasma urealyticum secretions, and herpes simplex virus type II secretion samples are divided into centrifuge tubes at 165 μL per tube, and labeled as N1, N2, N3, N4, N5, N6, N7, N8)

[0101] 2. 8 samples to be tested are positive (Xiamen Hospital of Traditional Chinese Medicine) (P1, P2, P3, P4, P5, P6, P7, P8) (Clinically collect urine positive samples, the concentration is P11.0×10 7 copies / mL, P21.0×10 6 copies / mL, P31.0×10 5 copies / mL, P41.0×10 5 copies / mL, P51.0×10 4 copies / mL, P61.0×10 4 copies / mL, p71.0×10 3 copies / mL), p81.0×10 3 copies / mL).

[0102] ...

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Abstract

The invention provides a human cytomegalovirus nucleic acid quantitative detection primer and probe, a kit and application thereof. The primer has a sequence shown as SEQ ID NO:1 and SEQ ID NO:2; the probe has a sequence shown as SEQ ID NO:4, and a 5'-termination single strand base sequence refers to 5-10 bases complementary to a target sequence and the middle 13-20 bases; a 3'-termination sequence refers to a sequence with 5-10 bases; the 5'-termination single strand base sequence and a 3'-termination sequence are partially matched to form a neck structure; the probe has a neck ring structure; the TM value is equal to (neck ring TM value+7)+ / -1; 18-25 bases are arranged at the 5'-termination and can be completely matched with target genes, totaling about 23-32bp; fluorescent and non-fluorescent quenching groups are respectively labeled at the 5'-termination and 3'-termination. The primer and probe have a stable linear structure, are used for quantitatively detecting human cytomegalovirus nucleic acid and have the advantages of low fluorescent background value, high signal to noise ratio and high detection sensitivity.

Description

Technical field [0001] The present invention relates to in vitro diagnostic reagents. In particular, it relates to a primer and probe for quantitative detection of human cytomegalovirus nucleic acid, a kit and its use. Background technique [0002] Human Cytomegalovirus (HCMV) belongs to the herpes subfamily Group B of the Herpesvirus family. Existing human cytomegalovirus nucleic acid quantitative detection methods have a series of problems. For example, although electron microscopy technology is direct and objective, it requires high virus titer, expensive equipment, and requires skilled technicians, so the detection value is low. Virus isolation is not easy to carry out in large quantities due to the high technical requirements, long isolation time, and easy contamination. The detection of CMV-DNA by conventional PCR is simple and fast. However, since the amplified product needs to be detected by electrophoresis, it is easy to cause cross contamination and false positive re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6806C12Q1/686C12Q1/70C12Q2531/113C12Q2525/301C12Q2563/107C12Q2523/308
Inventor 魏超林家旺魏劭
Owner 厦门安普利生物工程有限公司
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