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Primer and method for detecting HTLV (human T-lymphotropic virus)-I and HTLV-II proviruses in same pipe

A provirus and tube detection technology, applied in the field of medical testing, can solve the problems of false positives, false negatives, and poor specificity, and achieve the effects of high specificity, high accuracy, and short detection cycle

Active Publication Date: 2014-07-02
SHANGHAI ADICON CLINICAL LAB LNC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have shown that not all HTLV-infected patients will produce antibodies, and there are still single-cell infections in blood samples, so there are some false positive and false negative problems in serological testing, and the specificity is poor, which cannot solve the above problems well

Method used

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  • Primer and method for detecting HTLV (human T-lymphotropic virus)-I and HTLV-II proviruses in same pipe
  • Primer and method for detecting HTLV (human T-lymphotropic virus)-I and HTLV-II proviruses in same pipe
  • Primer and method for detecting HTLV (human T-lymphotropic virus)-I and HTLV-II proviruses in same pipe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 A primer and kit for detecting HTLV-I and HTLV-II proviruses in one tube

[0040] Primers for detecting HTLV-I and HTLV-II provirus, including specific primers (SEQ NO1 and SEQ NO2) and probe (SEQ NO3) for detecting HTLV-I provirus and specificity for detecting HTLV-II provirus Primers (SEQ NO4 and SEQ NO5) and probe (SEQ NO6), wherein:

[0041] (i) Specific primers and probes for detecting HTLV-I provirus:

[0042] SEQ NO1: CCCATTGGCTCCTGTGAAAG;

[0043] SEQ NO2: TTGAGACAGCGCCACGAAC;

[0044] SEQ NO 3: JOE-TCTCGGACTTTTGCATGGCCTCTCC-TAMARA

[0045] (ii) Specific primers and probes for detecting HTLV-II provirus:

[0046] SEQ NO4: TTGCCATACCTGTCAAACCATC;

[0047] SEQ NO5: CTGAAGAACGGCGCTGATAGTC;

[0048] SEQ NO 6: FAM-CTTCTCCGGTGCGGTTTCCGTCT-TAMARA.

[0049] The above two primers and probes were added to the same PCR tube, and the samples were detected by Real-time PCR reaction conditions.

[0050] A kit for detecting HTLV-I and HTLV-II proviruses, includ...

Embodiment 2

[0059] Example 2 A method for detecting HTLV-I and HTLV-II proviruses in one tube

[0060] A method for the simultaneous detection of HTLV-I and HTLV-II proviruses comprises:

[0061] (1) extract the DNA in the sample;

[0062] (2) Using the DNA in step (1) as a template, add primers and probes for detecting HTLV-I provirus and primers and probes for detecting HTLV-II provirus in the same PCR tube at a reasonable concentration ratio, And the sample is detected by Real-time PCR reaction, wherein:

[0063] (i) Specific primers and probes for detecting HTLV-I provirus:

[0064] SEQ NO1: CCCATTGGCTCCTGTGAAAG;

[0065] SEQ NO2: TTGAGACAGCGCCACGAAC;

[0066] SEQ NO 3: JOE-TCTCGGACTTTTGCATGGCCTCTCC-TAMARA

[0067] (ii) Specific primers and probes for detecting HTLV-II provirus:

[0068] SEQ NO4: TTGCCATACCTGTCAAACCATC;

[0069] SEQ NO5: CTGAAGAACGGCGCTGATAGTC;

[0070] SEQ NO 6: FAM-CTTCTCCGGTGCGGTTTCCGTCT-TAMARA.

[0071] (3) According to the results of Real-time PCR to ana...

Embodiment 3

[0078] Embodiment 3: Sensitivity determination

[0079] Respectively, the recombinant HTLV-I plasmid and HTLV-II plasmid were diluted in gradient (plasmid gradient was 10, 10 2 、10 3 、10 4 、10 5 、10 6 copies / ul) as a template, according to the method of the present invention for Real-time PCR reaction. figure 1 It is the two standard curves generated by the amplification of the real-time PCR detection system in the same tube (the plasmid gradient is 10, 10 2 、10 3 、10 4 、10 5 、10 6 copies / ul), where the HTLV-I standard curve Slope is -3.41, R 2 is 0.996; HTLV-II standard curve Slope is -3.35, R 2 is 0.995. According to Slope and R 2 The values ​​show that the amplification effect of the same-tube Real-time system of the present invention is ideal, and the lowest limit can reach 10 copies / ul.

[0080] figure 2 It is the result of amplifying two kinds of 10copies / ul HTLV-I plasmid and HTLV-II plasmid by the real-time PCR detection system in the same tube.

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Abstract

The invention provides a primer and a method for detecting HTLV (human T-lymphotropic virus)-I and HTLV-II proviruses. The primer comprises a specific primer and a probe for detecting an HTLV-I provirus and a specific primer and a probe for detecting an HTLV-II provirus, the two primers and the two probes are added in the same PCR (polymerase chain reaction) pipe according to a reasonable concentration ratio, and a sample is detected by means of optimized Real-time PCR reaction conditions. The detection method can be used for detecting whether HTLV infection exists before serological changes, thereby greatly shortening the window phase; meanwhile, compared with a conventional serological detection method, the detection method has the advantages of short detection period, high specificity, high accuracy, high sensitivity, little dependence on the conditions, low pollution risk and the like. The detection result is beneficial to monitor the change of the carrying capacity of the HTLV provirus of a patient and limit the propagation of the HTLV virus.

Description

technical field [0001] The invention belongs to the field of medical testing, in particular to a primer and a method for detecting HTLV-I and HTLV-II proviruses in one tube. Background technique [0002] Human T-cell lymphotropic virus (HTLV) is the first human retrovirus discovered by American Gallo in 1980 from patients with cutaneous T-cell lymphoma. Divided into type 1 (HTLV-I) and type 2 (HTLV-II). The virus is a spherical particle, the interior is composed of RNA nucleoprotein and the surrounding 20-hedron protein capsid, the outermost layer has an envelope structure, and the surface is embedded with glycoproteins. At present, about 10 to 20 million people in the world are infected with HTLV virus, and HTLV infection cases have been found in more than 10 provinces and cities in my country, including Beijing, Guangxi, Jiangxi, Xinjiang, Liuzhou, Hefei and Sichuan, and some coastal areas have also appeared local small-scale epidemic. [0003] HTLV infects cells mai...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2547/101C12Q2561/113
Inventor 陈奕磊黎洪奋王淑一
Owner SHANGHAI ADICON CLINICAL LAB LNC
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