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Primers, probe, kit and detection method for type II nucleic acid quantitative detection of herpes simplex virus

A herpes simplex virus, detection kit technology, applied in biochemical equipment and methods, microorganism-based methods, and microbial determination/inspection, etc., can solve the problems of false positives and high probe TM values

Inactive Publication Date: 2014-05-21
厦门安普利生物工程有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the high GC content in the conserved region of the HSV II genome, the key technology for applying fluorescent quantitative PCR technology to HSV II-DNA detection is the design of primers and fluorescent probes. The TM value of the probe is too high due to the high GC content of ordinary probe design. Prone to non-specific binding, leading to false positive results

Method used

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  • Primers, probe, kit and detection method for type II nucleic acid quantitative detection of herpes simplex virus
  • Primers, probe, kit and detection method for type II nucleic acid quantitative detection of herpes simplex virus
  • Primers, probe, kit and detection method for type II nucleic acid quantitative detection of herpes simplex virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: the preparation of kit

[0070] Components of various reagents:

[0071] Lysate: (10mmMPH7.0) (sigma), silica (1‰), Triton X-100 (1‰), EDTA·2Na (10mM), guanidine isothiocyanate (2M);

[0072] detergent: (10mmMPH7.0), Triton X-100 (1‰), EDTA·2Na (1mM), guanidine isothiocyanate (2M);

[0073] Repeat wash solution: (10mmMPH7.0), Triton X-100 (1‰), EDTA·2Na (1mM)

[0074] HSVⅡPCR reaction tube:

[0075] Taq enzyme (2U), UNG enzyme (0.01U), EDTA·2Na (0.1mM), paraffin oil (25 microliters);

[0076] HSVⅡ reaction buffer: HSVⅡ upstream and downstream primers (10pM), HSVⅡ probe (5pM), (20Mmph9.0), KCl (50mM), MgCl2 (1.5mM), EDTA·2Na (0.1mM), dNtps (0.2mM);

[0077] HSVⅡquantitative standards are shown in Table 2

[0078]

[0079] HSVⅡV negative control: normal saline;

[0080] HSVⅡ critical positive control: normal saline, virus-like particles;

[0081] HSVⅡ strong positive control: normal saline, virus-like particles.

[0082] The preparation me...

Embodiment 2

[0089] Example 2: Detection of samples using kits

[0090] (1) Sample

[0091] 1. 8 samples were negative

[0092] Clinical collection of HPV6 type secretion samples, HPV11 type vaginal discharge samples, HPV16 type vaginal discharge samples, HPV18 type vaginal discharge samples, Chlamydia trachomatis secretion samples, Neisseria gonorrhoeae secretion samples, Ureaplasma urealyticum secretion samples Samples, human cytomegalovirus urine samples, divided into centrifuge tubes at 165 μL / cartridge, labeled as N1, N2, N3, N4, N5, N6, N7, N8 in turn. (Xiamen Hospital of Traditional Chinese Medicine)

[0093] 2. 8 samples to be tested are positive (clinical collection of herpes simplex virus type II secretion 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). (Xiamen Hospital of Traditional Chinese Medicine)

[0094]...

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Abstract

The invention provides primers, a probe, a kit and a detection method for type II nucleic acid quantitative detection of herpes simplex virusherpes simplex virus. The primers are as shown in the sequences of SEQ ID NO: 1 and SEQ ID NO: 2; the probe is as shown in the sequence of SEQ ID NO: 4, and a reporter fluorescent group and a non-quenching fluorescent group are marked at the two ends of the probe, respectively; the kit comprises the primers and the probe. The primers, the probe and the kit thereof are low in fluorescent background value, high in signal-to-noise ratio and high in detection sensitivity when used for detecting type II nucleic acid quantitation of the herpes simplex virus.

Description

technical field [0001] The present invention relates to in vitro diagnostic reagents. In particular, it relates to a primer and probe, a kit and a detection method for the quantitative detection of herpes simplex virus type II nucleic acid. Background technique [0002] Herpes simplex virus Ⅱ (herpes simplex virus Ⅱ, HSV Ⅱ) is one of the common pathogens in human viral diseases. Herpes simplex virus type II (HSV II) detection methods have a series of problems, eg. Although herpes simplex virus is easy to carry out in vitro cell culture and can produce visible cell lesions, the detection value of virus isolation and culture is low due to high technical requirements, long separation time, easy pollution, expensive equipment, and skilled technicians. Not conducive to large-scale development. Conventional PCR detection of HSVⅡ-DNA is simple and fast. However, since the amplified product needs to be detected by electrophoresis, it is easy to cause false positive results due t...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/70C12Q1/686
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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