Kit for detecting human cytomegalovirus based on fluorescence PCR or mass spectrometry

Abstract

The invention discloses a kit for detecting human cytomegalovirus based on fluorescent PCR or mass spectrometry. The invention provides a primer probe set. The primer probe set is composed of a primerHCMV-F shown in SEQ ID NO: 1, a primer HCMV-R shown in SEQ ID NO: 2 and a probe HCMV-P1. The nucleotide of the probe HCMV-P1 is as shown in SEQ ID NO: 3. The invention further provides the primer probe set. The primer probe set is composed of the primer HCMV-F shown in SEQ ID NO: 1, the primer HCMV-R shown in SEQ ID NO: 2 and the probe HCMV-P2 shown in SEQ ID NO: 11. The invention provides two kits. Core elements of the kit are specific primer probe groups, one kit is used for detection based on a fluorescent quantitative PCR method, the other kit is used for detection based on a nucleic acidmass spectrometry method, qualitative or quantitative detection of HCMV nucleic acid can be realized, and the kit has the advantages of high sensitivity, high specificity, high accuracy, simplicity,convenience and quickness.

Description

technical field

[0001] The invention relates to a kit for detecting human cytomegalovirus based on fluorescent PCR or mass spectrometry. Background technique

[0002] Human cytomegalovirus (Human Cytomegalic Virus, HCMV) belongs to the herpesviridae family and is the largest virus in the human herpesvirus group, with a diameter of 100-200nm and a core of double-stranded linear DNA, which has a typical herpesvirus structure. HCMV can only infect humans and proliferate in human fibroblasts. HCMV proliferates slowly in cell culture and has a long replication cycle. It takes 30 to 40 days for the initial isolation and culture to appear cytopathic changes, which are characterized by enlarged and rounded cells, enlarged nuclei, and large cytotropic cells surrounded by a "halo" in the nucleus. Acidic inclusion bodies.

[0003] HCMV infection is very widespread in the population. The infection rate of adults in China is over 95%. It is usually a recessive infection. Most infected ...

Examples

Embodiment 1

[0061] Embodiment 1, the preparation of fluorescent PCR kit

[0062] 1. Design the primer-probe set

[0063] After the design and screening of primers and probes, and the comparison and verification of the effects, the primer-probe set A and the primer-probe set B were obtained.

[0064] The target gene of primer probe set A is DNA polymerase gene. Primer probe set A consists of HCMV-F (upstream primer), HCMV-R (downstream primer) and HCMV-P1 (probe).

[0065] HCMV-F (SEQ ID NO: 1): 5'-cacctaacgctgctatcatctc-3';

[0066] HCMV-R (SEQ ID NO: 2): 5'-ctgactggacggcagattg-3';

[0067] HCMV-P1 (SEQ ID NO: 3): 5'-FAM-tatgtttcagatgtcgccgccctt-BHQ-3'.

[0068] The target gene of primer probe set B is the internal standard gene (also known as IC gene, which is an artificially constructed gene). Primer-probe set B consists of IC-F (upstream primer), IC-R (downstream primer) and IC-P1 (probe).

[0069] IC-F (SEQ ID NO: 4): 5'-CCTCACAATACCACAGAGTC-3';

[0070] IC-R (SEQ ID NO: 5): 5'...

Embodiment 2

[0120] Embodiment 2, the performance of kit

[0121] 1. The minimum detection limit of the qualitative test

[0122] 1. Get the standard plasmid HCMV prepared in step 2 of Example 1 and dilute with TE buffer to obtain a concentration of 1.0×10 4 copies / ml or 1.0×10 3 copies / ml of template DNA solution.

[0123] 2. Perform fluorescent PCR to obtain the Ct value.

[0124] Reaction system (30 μl): 20 μl of HCMV reaction premix solution prepared in step 3 of Example 1+10 μl of template DNA solution.

[0125] Reaction program: 50°C for 2min; 95°C for 10min; 95°C for 15sec, 64°C for 60sec, 45 cycles (fluorescence signal was collected at 64°C).

[0126] Carry out 10 repeated tests, and the Ct value results of the HCMV detection channel are shown in Table 2.

[0127] Table 2

[0128] 1 2 3 4 5 6 7 8 9 10 1.0×10 4 copies / ml

34.55 35.30 35.01 36.98 35.75 36.42 35.88 36.71 35.67 36.54 1.0×10 3 copies / ml

37.25 No Ct value 38.56 39...

Embodiment 3

[0183] Embodiment 3, detect by mass spectrometry

[0184] 1. Design the primer-probe set

[0185] Primer probe set C consists of HCMV-F (upstream primer), HCMV-R (downstream primer) and HCMV-P2 (probe).

[0186] HCMV-P2 (SEQ ID NO: 11): 5'-tatgtttcagatgtcgccgccct-3'.

[0187] The primer-probe set consists of IC-F (upstream primer), IC-R (downstream primer) and IC-P2 (probe).

[0188] IC-P2 (SEQ ID NO: 12): 5'-tctgccaagtgtttgctgacg-3'.

[0189] Primer-probe set IE1-2 consists of IE1-F (upstream primer), IE1-R (downstream primer) and IE1-P2 (probe).

[0190] IE1-P2 (SEQ ID NO: 13): 5'-agacggaagagaaattcactggcg-3'.

[0191] The primer-probe set PP-2 consists of PP-F (upstream primer), PP-R (downstream primer) and PP-P2 (probe).

[0192] PP-P2 (SEQ ID NO: 14): 5'-acgatgccgagatcgtggaac-3'.

[0193] Compared with the probe of Example 1, the difference in the nucleotide sequence of the probe of this example is only one nucleotide at the 3' end, which is used to distinguish wheth...

technical field

[0001] The invention relates to a kit for detecting human cytomegalovirus based on fluorescent PCR or mass spectrometry. Background technique

[0002] Human cytomegalovirus (Human Cytomegalic Virus, HCMV) belongs to the herpesviridae family and is the largest virus in the human herpesvirus group, with a diameter of 100-200nm and a core of double-stranded linear DNA, which has a typical herpesvirus structure. HCMV can only infect humans and proliferate in human fibroblasts. HCMV proliferates slowly in cell culture and has a long replication cycle. It takes 30 to 40 days for the initial isolation and culture to appear cytopathic changes, which are characterized by enlarged and rounded cells, enlarged nuclei, and large cytotropic cells surrounded by a "halo" in the nucleus. Acidic inclusion bodies.

[0003] HCMV infection is very widespread in the population. The infection rate of adults in China is over 95%. It is usually a recessive infection. Most infected ...

Examples

Embodiment 1

[0061] Embodiment 1, the preparation of fluorescent PCR kit

[0062] 1. Design the primer-probe set

[0063] After the design and screening of primers and probes, and the comparison and verification of the effects, the primer-probe set A and the primer-probe set B were obtained.

[0064] The target gene of primer probe set A is DNA polymerase gene. Primer probe set A consists of HCMV-F (upstream primer), HCMV-R (downstream primer) and HCMV-P1 (probe).

[0065] HCMV-F (SEQ ID NO: 1): 5'-cacctaacgctgctatcatctc-3';

[0066] HCMV-R (SEQ ID NO: 2): 5'-ctgactggacggcagattg-3';

[0067] HCMV-P1 (SEQ ID NO: 3): 5'-FAM-tatgtttcagatgtcgccgccctt-BHQ-3'.

[0068] The target gene of primer probe set B is the internal standard gene (also known as IC gene, which is an artificially constructed gene). Primer-probe set B consists of IC-F (upstream primer), IC-R (downstream primer) and IC-P1 (probe).

[0069] IC-F (SEQ ID NO: 4): 5'-CCTCACAATACCACAGAGTC-3';

[0070] IC-R (SEQ ID NO: 5): 5'...

Embodiment 2

[0120] Embodiment 2, the performance of kit

[0121] 1. The minimum detection limit of the qualitative test

[0122] 1. Get the standard plasmid HCMV prepared in step 2 of Example 1 and dilute with TE buffer to obtain a concentration of 1.0×10 4 copies / ml or 1.0×10 3 copies / ml of template DNA solution.

[0123] 2. Perform fluorescent PCR to obtain the Ct value.

[0124] Reaction system (30 μl): 20 μl of HCMV reaction premix solution prepared in step 3 of Example 1+10 μl of template DNA solution.

[0125] Reaction program: 50°C for 2min; 95°C for 10min; 95°C for 15sec, 64°C for 60sec, 45 cycles (fluorescence signal was collected at 64°C).

[0126] Carry out 10 repeated tests, and the Ct value results of the HCMV detection channel are shown in Table 2.

[0127] Table 2

[0128] 1 2 3 4 5 6 7 8 9 10 1.0×10 4 copies / ml

34.55 35.30 35.01 36.98 35.75 36.42 35.88 36.71 35.67 36.54 1.0×10 3 copies / ml

37.25 No Ct value 38.56 39...

Embodiment 3

[0183] Embodiment 3, detect by mass spectrometry

[0184] 1. Design the primer-probe set

[0185] Primer probe set C consists of HCMV-F (upstream primer), HCMV-R (downstream primer) and HCMV-P2 (probe).

[0186] HCMV-P2 (SEQ ID NO: 11): 5'-tatgtttcagatgtcgccgccct-3'.

[0187] The primer-probe set consists of IC-F (upstream primer), IC-R (downstream primer) and IC-P2 (probe).

[0188] IC-P2 (SEQ ID NO: 12): 5'-tctgccaagtgtttgctgacg-3'.

[0189] Primer-probe set IE1-2 consists of IE1-F (upstream primer), IE1-R (downstream primer) and IE1-P2 (probe).

[0190] IE1-P2 (SEQ ID NO: 13): 5'-agacggaagagaaattcactggcg-3'.

[0191] The primer-probe set PP-2 consists of PP-F (upstream primer), PP-R (downstream primer) and PP-P2 (probe).

[0192] PP-P2 (SEQ ID NO: 14): 5'-acgatgccgagatcgtggaac-3'.

[0193] Compared with the probe of Example 1, the difference in the nucleotide sequence of the probe of this example is only one nucleotide at the 3' end, which is used to distinguish wheth...

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