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Specific primer and probe for detection of HTLV-I and HTLV-II and fluorescent quantitative PCR detection kit

A real-time fluorescence quantitative and specific technology, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of unsuitable blood screening, increased fluorescence intensity, poor specificity, etc., to reduce economic burden, Effects of reducing workload and relieving pain

Inactive Publication Date: 2018-09-04
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescent dye intercalation technology uses SYBRgreen I to intercalate DNA double-strands to increase the fluorescence intensity. The fluorescent signal is introduced into double-stranded DNA. This method has poor specificity, and the results are often interfered by the presence of primer dimers, resulting in false positives, etc. problems and therefore not suitable for blood screening

Method used

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  • Specific primer and probe for detection of HTLV-I and HTLV-II and fluorescent quantitative PCR detection kit
  • Specific primer and probe for detection of HTLV-I and HTLV-II and fluorescent quantitative PCR detection kit
  • Specific primer and probe for detection of HTLV-I and HTLV-II and fluorescent quantitative PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 LTR gene standard product

[0028] To establish a real-time fluorescent quantitative PCR method, the external standard required by the method must first be prepared. The standard should contain highly conserved and specific sequences, and high specificity of the reaction must be ensured.

[0029] Under the electron microscope, HTLV-1 and HTLV-2 are spherical, with a diameter of about 100nm. The center is the viral RNA and reverse transcriptase. The outermost layer is the envelope of the virus. The surface is embedded with gp120, which can bind to CD4 to mediate the virus Infect. Inside the envelope is the capsid of the virus, which contains two structural proteins, P18 and P24. The genome of the virus consists of three structural genes, gag, pol, and env, and two regulatory genes, tax and rex, from the 5' to the 3' end, both ends of which are LTRs.

[0030] However, HTLV-1 and HTLV-2 have about 70% homology in genetics. Therefore, the s...

Embodiment 2

[0084] Embodiment 2 real-time fluorescent quantitative PCR kit

[0085] 1. Design and synthesis of specific primers and probes

[0086] Taking the conserved fragment of the LTR rDNA gene of HTLV selected in Example 1 as the target, a set of real-time fluorescent quantitative PCR primers and probes were designed using PrimerPremier 5 software and Beacon Designer 8.

[0087] As the core of the present invention, a group of primers and probe nucleotide sequences and amplified fragments for HTLV real-time fluorescent PCR detection are as follows:

[0088] Upstream primer: 5'-CCTTATATCAGAGGCCGAA-3';

[0089] Downstream primer: 5'-TCTGGCAGCCCATTGTCAA-3'.

[0090] Probe: 5'-FAM-AGTGCCAAAGACCCTTCCTGGGCCTCT-TAMRA-3'.

[0091] The fluorescent reporter group at the 5' end of the probe is FAM, and the fluorescent quencher group TAMRA is labeled at the 3' end. At the same time, other fluorescent reporter groups such as TET, JOE, HEX, VIC and fluorescent quencher groups such as DABCYL, BH...

Embodiment 3

[0114] Example 3 The quantitative detection method of the sample to be tested by the real-time fluorescent quantitative PCR kit

[0115] Respectively with the sample DNA to be tested and the pMD18-T-LTR rDNA series concentration standard (1.00 × 10 7 copies / ml, 1.00×10 6 copies / ml, 1.00×10 5 copies / ml, 1.00×10 4 copies / ml, 1.00×10 3 copies / ml) as a template, use the primers and probes in the kit to perform fluorescent quantitative PCR amplification, and set up positive and negative controls at the same time.

[0116] The PCR reaction system is as follows:

[0117]

[0118] Reaction conditions: 94°C pre-denaturation for 2 minutes, 94°C for 15 seconds, 60°C for 40s and collecting fluorescence signals, 40 cycles. Rapid quantitative detection through the standard curve and the Ct value of the sample to be tested.

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Abstract

The invention provides a specific primer and probe for simultaneous detection of HTLV-I and HTLV-II. The probe sequence is 5'-AGTGCCAAAGACCCTTCCTGGGCCTCT-3'. The specific primer is the following sequence or the complementary chain sequence of the following sequence, the upstream primer sequence is 5'-CCTTATATCAGAGGCCGAA-3', and the downstream primer sequence is 5'-TCTGGCAGCCCATTGTCAA-3'. Accordingto the specific primer and the probe, by extracting DNA in a to-be-detected sample and by combining the specific primer and the probe and a real-time fluorescent quantitative PCR detection technique,the purpose that HTLV-I and HTLV-II in the to-be-detected sample are accurately quantification can be achieved; and the infection states of HTLV-I and HTLV-II can be detected simultaneously only through one sample, and the specific primer and the probe are suitable for large-scale blood screening work in both cost and efficiency.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and in vitro diagnostic reagents, in particular to specific primers and probes and a fluorescent quantitative PCR detection kit for detecting HTLV-I and HTLV-II. Background technique [0002] Human T-lymphoblastic leukemia virus is the earliest discovered human retrovirus, including four subtypes. Among them, HTLV-1 and HTLV-2 infections are more common, and they are pathogens that cause adult T-cell leukemia and lymphoma, respectively. HTLV-3 and HTLV-4 infections are less common. HTLV-1 and HTLV-2 can be transmitted through vertical routes, sexual contact, parenteral blood transmission, and intravenous drug use, and are prevalent all over the world. my country belongs to the non-endemic area of ​​HTLV, but since 1984, infected persons have been found successively. In 2004, Xiamen, located in a high-endemic area, became the first city in my country to conduct HTLV-1 / 2 screening for b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/702C12Q1/6851C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/113
Inventor 吴玲车团结徐红陈游王晓燕李亚鹏
Owner SUZHOU BAIYUAN GENT CO LTD
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