Primers, probes and kit for synchronously detecting human herpesvirus-6 and human herpesvirus-7

Abstract

The invention discloses primers, probes and kit for synchronously detecting human herpesvirus-6 and human herpesvirus-7. According to the primers, the probes and the kit, through selecting a novel target gene and redesigning the primers and the probes, the reaction system is optimized, the false positive incidence of clinical synchronous detection on human herpesviruses is substantially lowered, the sensitivity is high, the specificity is good, and the time for detection is shorter, so that the clinical application and popularization of the technology can be achieved.

Description

technical field

[0001] The invention relates to the technical field of herpes virus detection, in particular to a primer, a probe and a kit for synchronous detection of human herpes virus types 6 and 7. Background technique

[0002] Existing studies have confirmed that human herpesvirus (human herpesvirus, HHV)-6, -7, -8 can be transmitted through blood transfusion, and invade human peripheral blood mononucleated cells CD4 + T cells, which subsequently enter a latent state and cause persistent infection, are activated to cause disease in immunocompromised and neurologically diseased individuals.

[0003] For herpes virus types 6, 7, and 8, a multiplex fluorescent quantitative PCR method is provided in the prior art, and U67, U36, and ORF65 are selected as target genes of HHV-6, HHV-7, and HHV-8 respectively. The above three viruses can be detected at one time, and the sensitivity is high, but its defect is that the false positive rate is high in use, especially for HHV-6 an...

Examples

Embodiment 1

[0033] The present embodiment provides a set of primers and fluorescent probes for simultaneous detection of human herpesvirus 6 and 7. The reorganization contains two pairs of primers and two probes. The sequence is as follows:

[0034] (1) Forward primer HHV6-F, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0035] Reverse primer HHV6-R, the nucleotide sequence of which is shown in SEQ ID NO.2;

[0036] Fluorescent probe HHV6-P, the nucleotide sequence of which is shown in SEQ ID NO.3, the 5' end of the fluorescent probe is labeled with FAM, and the 3' end quencher group BHQ;

[0037] (2) Forward primer HHV7-F, the nucleotide sequence of which is shown in SEQ ID NO.4;

[0038] Reverse primer HHV7-R, the nucleotide sequence of which is shown in SEQ ID NO.5;

[0039] Fluorescent probe HHV7-P, the nucleotide sequence of which is shown in SEQ ID NO.6, the 5' end of the fluorescent probe is labeled JOE, and the 3' end quencher group BHQ;

[0040] The above primers...

Embodiment 2

[0042] This embodiment provides a fluorescent quantitative PCR kit for simultaneous detection of human herpesvirus 6 and 7

[0043] 1. Kit composition and preparation

[0044] 1) Primer and fluorescent probe set

[0045] Same as Example 1

[0046] 2) DNA extraction solution

[0047] The DNA extraction solution can use a commercially available DNA extraction kit, or it can be prepared by itself.

[0048] In this example, the formula of the DNA extract solution: prepare 200mmol / L NaCl, 100mmol / L Tris-HCl, 1% SDS, 50mmol / L EDTA, adjust the pH to 8.5, 3mg / ml proteinase K (add before use)

[0049] 3) The reaction system of double fluorescent quantitative PCR is as follows:

[0050] Reagent

Final concentration or content per unit volume

PCR 10×buffer

5μl

MgCl 2

4mmol / L

dNTP (including dUTP)

0.48mmol / L

Tag DNA polymerase

0.5U

UNGase

0.2U

HHV-6 primer

0.29μmol / L

HHV-6 probe

0.24μmol / L

...

technical field

[0001] The invention relates to the technical field of herpes virus detection, in particular to a primer, a probe and a kit for synchronous detection of human herpes virus types 6 and 7. Background technique

[0002] Existing studies have confirmed that human herpesvirus (human herpesvirus, HHV)-6, -7, -8 can be transmitted through blood transfusion, and invade human peripheral blood mononucleated cells CD4 + T cells, which subsequently enter a latent state and cause persistent infection, are activated to cause disease in immunocompromised and neurologically diseased individuals.

[0003] For herpes virus types 6, 7, and 8, a multiplex fluorescent quantitative PCR method is provided in the prior art, and U67, U36, and ORF65 are selected as target genes of HHV-6, HHV-7, and HHV-8 respectively. The above three viruses can be detected at one time, and the sensitivity is high, but its defect is that the false positive rate is high in use, especially for HHV-6 an...

Examples

Embodiment 1

[0033] The present embodiment provides a set of primers and fluorescent probes for simultaneous detection of human herpesvirus 6 and 7. The reorganization contains two pairs of primers and two probes. The sequence is as follows:

[0034] (1) Forward primer HHV6-F, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0035] Reverse primer HHV6-R, the nucleotide sequence of which is shown in SEQ ID NO.2;

[0036] Fluorescent probe HHV6-P, the nucleotide sequence of which is shown in SEQ ID NO.3, the 5' end of the fluorescent probe is labeled with FAM, and the 3' end quencher group BHQ;

[0037] (2) Forward primer HHV7-F, the nucleotide sequence of which is shown in SEQ ID NO.4;

[0038] Reverse primer HHV7-R, the nucleotide sequence of which is shown in SEQ ID NO.5;

[0039] Fluorescent probe HHV7-P, the nucleotide sequence of which is shown in SEQ ID NO.6, the 5' end of the fluorescent probe is labeled JOE, and the 3' end quencher group BHQ;

[0040] The above primers...

Embodiment 2

[0042] This embodiment provides a fluorescent quantitative PCR kit for simultaneous detection of human herpesvirus 6 and 7

[0043] 1. Kit composition and preparation

[0044] 1) Primer and fluorescent probe set

[0045] Same as Example 1

[0046] 2) DNA extraction solution

[0047] The DNA extraction solution can use a commercially available DNA extraction kit, or it can be prepared by itself.

[0048] In this example, the formula of the DNA extract solution: prepare 200mmol / L NaCl, 100mmol / L Tris-HCl, 1% SDS, 50mmol / L EDTA, adjust the pH to 8.5, 3mg / ml proteinase K (add before use)

[0049] 3) The reaction system of double fluorescent quantitative PCR is as follows:

[0050] Reagent

Final concentration or content per unit volume

PCR 10×buffer

5μl

MgCl 2

4mmol / L

dNTP (including dUTP)

0.48mmol / L

Tag DNA polymerase

0.5U

UNGase

0.2U

HHV-6 primer

0.29μmol / L

HHV-6 probe

0.24μmol / L

...

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