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Primers, probes, kit and method for synchronous amplification detection of HCMV, HSV1, HSV2 and B19

A kit and probe technology, which can be used in microorganism-based methods, biochemical equipment and methods, introduction of foreign genetic material using vectors, etc., and can solve problems such as unreported

Inactive Publication Date: 2017-06-13
东莞市儿童医院 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the multiplex PCR reaction technology for multiple viruses is quite mature, but there is no report on the multiplex fluorescent quantitative PCR method for these four viruses

Method used

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  • Primers, probes, kit and method for synchronous amplification detection of HCMV, HSV1, HSV2 and B19
  • Primers, probes, kit and method for synchronous amplification detection of HCMV, HSV1, HSV2 and B19
  • Primers, probes, kit and method for synchronous amplification detection of HCMV, HSV1, HSV2 and B19

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Preparation of standard curves for cytomegalovirus, herpes simplex virus types 1 and 2 and parvovirus B19 using kits.

[0085]Establishment of standard curve for multiple detection kits (take Agilent's mx3000p as an example)

[0086] Material:

[0087] a. 4 μL of 10×PCR buffer, 5U / μL Taq DNA polymerase 0.5 μL, 25mM MgCl 2 3.0 μL, 10 mM dNTPs 1.0 μL, 10 μmol / L HCMV, HSV1, HSV2 and B19 upstream and downstream primers 0.8 μL each, 10 μmol / L HCMV, HSV1, HSV2 and B19 probes 1.6 μL each, template DNA 2 μL, no Bacterial double distilled water 16.7μL, the total reaction solution is 40μL;

[0088] b. Standard positive template stock solution: the concentration is 10 7 Copy / μL standard positive template, and then perform 10-fold serial dilution;

[0089] c. Negative quality control standard: sterile double distilled water.

[0090] method:

[0091] A. Serially dilute the positive standard template to 10 7 copies / μL, 10 6 copies / μL, 10 5 copies / μL, 10 4 copie...

Embodiment 2

[0094] Example 2: An example of detection of clinical samples by a multiplex detection kit (taking Agilent's mx3000p as an example)

[0095] Take 38 μL of the fluorescent quantitative PCR reaction solution, 2 μL of the positive standard template, and set a negative control, respectively add to different PCR reaction tubes, and perform PCR detection in parallel on the fluorescent quantitative PCR instrument. The cycle conditions are: 50°C for 2min, 94°C for 30s, and the cycle process uses a two-step method of 94°C for 5s, 60°C for 1min, 40 cycles. Simultaneous detection of FAM, HEX, ROX and CY5. After the cycle is over, use the software that comes with the instrument to read the results. See Figure 6 , the amplification curves of the positive standard and clinical samples present a smooth S-shape, indicating that the amplification efficiency is good. Virus quantification results can be calculated according to the standard curve in Implementation 1.

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Abstract

The invention relates to the technical field of biological detection, and particularly relates to primers, probes, a kit and a method for synchronous amplification detection of HCMV, HSV1, HSV2 and B19; the primers include four pairs of primers, namely totally 8 primers, for amplifying an HCMV virus UL75 gene, an HSV1 virus UL12 gene, an HSV2 virus UL15 gene and a B19 virus VP1 gene; the probes include 4 probes for detecting the HCMV virus UL75 gene, the HSV1 virus UL12 gene, the HSV2 virus UL15 gene and the B19 virus VP1 gene; the kit comprises the primers and the probes. The primers, probes, kit and method have the advantages of accuracy, sensitivity, short window period, high efficiency and the like, the operation is relatively simple, and the incidence rate of mother and infant vertical transmission diseases can be reduced.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer, a probe, a kit and a method for synchronously amplifying and detecting HCMV, HSV1, HSV2 and B19. Background technique [0002] Cytomegalovirus (CMV), herpes simplex virus (HSV) types 1 and 2, and parvovirus B19 (B19) are four common human herpesviruses that cause congenital intrauterine infections and perinatal infections that cause perinatal malformations . After these pathogens infect the mother, the relevant symptoms of the mother are not obvious, but they can be transmitted vertically to the fetus through the placenta, causing serious sequelae to the fetus and newborn. The clinical symptoms of neonatal infection depend on the type of virus and the gestational age at the time of infection. The risk of fetal virus infection is usually negatively correlated with gestational age. After infection, neonatal pneumonia, hearing abnormalities, hyperbilirubinemi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12N15/64C12N15/70C12R1/19C12R1/93
CPCC12N15/64C12N15/70C12N2800/101C12Q1/6851C12Q1/701C12Q1/705C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/113C12Q2563/107
Inventor 黎四平马强李文瑞陆小梅钟柏茂张守涛
Owner 东莞市儿童医院
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