Probe, primer, detection kit and detection method for detecting arboviruses based on suspension microsphere array system

Abstract

The invention discloses a probe, a primer, a detection kit and a detection method for detecting arboviruses based on a suspension microsphere array system. According to the technical scheme, the probefor detecting the arboviruses is coupled with different coding microspheres, a suspension microsphere array is obtained after mixing, the specific primer provided by the invention is then used for carrying out multiple RT-PCR amplification on a sample to be detected, the PCR product is in hybridization with the suspension microsphere array, streptavidin R- phycoerythrin is then used for carryingout developing, then a suspension microsphere array reading instrument is used for reading the detection result, and that whether the sample to be detected contains the detected arboviruses or not isjudged. The probe and the primer for detecting the arboviruses based on the suspension microsphere array system can simultaneously detect 7 kinds of arboviruses, thus the detection time is greatly shortened, and the detection efficiency and accuracy are improved.

Description

Technical field:

[0001] The invention belongs to the field of molecular biology, and in particular relates to a probe, a primer, a detection kit and a detection method for detecting insect vector viruses based on a suspended microbead array system. Background technique:

[0002] Arth-borne viruses refer to a class of viruses that use blood-sucking insects as the medium to transmit a variety of serious diseases among vertebrates, humans and animals. In recent years, the dengue fever virus and Zika virus infections that have been prevalent in the tropical jungle areas of the world and in southern my country are all insect-borne viruses. Patients infected with arboviruses have the same clinical symptoms, including fever, muscle and joint pain, rash, swollen lymph nodes, and so on. At present, ELISA or fluorescent quantitative PCR method is mainly used for clinical diagnosis of a single virus, so there is a risk of missed diagnosis.

[0003] Some reports have made some attempt...

Examples

Embodiment 1

[0050] 1. Multiplex PCR primer design:

[0051] The above 7 viruses are all RNA viruses, so the multiple reverse transcription PCR of these 7 RNA viruses is designed to react in one tube. The PCR primer sequences corresponding to these 7 viruses and the human GAPDH gene used as internal reference are shown in Table 1 .

[0052] 5 of all reverse primers (i.e. primers GAPDH-R, DENV1-R, DENV2-R, DENV3-R, DENV4-R, NS2A-R, TBE-R, YFV-R, Chikv-R and Zika-R) ' end labeled with biotin. Part of the primer sequence contains degenerate bases, where Y stands for base C or T, R stands for base A or G, V stands for base G or A or C, N stands for base A or T or G or C, D Represents the base G or A or T, K represents the base G or T.

[0053] Table 1. Multiplex PCR primer sequences for detection of RNA viruses

[0054]

[0055] Note: F means forward primer; R means reverse primer

[0056] 2. Probe design:

[0057] The present invention designs a total of 12 probes for 7 kinds of inse...

Embodiment 2

[0091] According to the method of Example 1, 10 positive clinical samples confirmed to be dengue virus type 1 infection by antigen method and PCR method were detected, and the positive detection rate results are shown in Table 5.

[0092] Table 5. The positive detection rate of dengue virus type 1 by optimized primers

[0093]

[0094] It can be seen from Table 5 that after the degenerate base design of the primers, the positive detection rate is significantly improved, and positive samples can be detected to the greatest extent.

Embodiment 3

[0096] A pregnant woman with fever and muscle soreness symptoms is detected to be dengue virus positive with the dengue antigen method, but it is not known to be the dengue virus of that type, and it is determined to be dengue type 2 virus according to the method of embodiment 1 (see figure 1 ), the result was the same as the antigen method and PCR method.

Technical field:

[0001] The invention belongs to the field of molecular biology, and in particular relates to a probe, a primer, a detection kit and a detection method for detecting insect vector viruses based on a suspended microbead array system. Background technique:

[0002] Arth-borne viruses refer to a class of viruses that use blood-sucking insects as the medium to transmit a variety of serious diseases among vertebrates, humans and animals. In recent years, the dengue fever virus and Zika virus infections that have been prevalent in the tropical jungle areas of the world and in southern my country are all insect-borne viruses. Patients infected with arboviruses have the same clinical symptoms, including fever, muscle and joint pain, rash, swollen lymph nodes, and so on. At present, ELISA or fluorescent quantitative PCR method is mainly used for clinical diagnosis of a single virus, so there is a risk of missed diagnosis.

[0003] Some reports have made some attempt...

Examples

Embodiment 1

[0050] 1. Multiplex PCR primer design:

[0051] The above 7 viruses are all RNA viruses, so the multiple reverse transcription PCR of these 7 RNA viruses is designed to react in one tube. The PCR primer sequences corresponding to these 7 viruses and the human GAPDH gene used as internal reference are shown in Table 1 .

[0052] 5 of all reverse primers (i.e. primers GAPDH-R, DENV1-R, DENV2-R, DENV3-R, DENV4-R, NS2A-R, TBE-R, YFV-R, Chikv-R and Zika-R) ' end labeled with biotin. Part of the primer sequence contains degenerate bases, where Y stands for base C or T, R stands for base A or G, V stands for base G or A or C, N stands for base A or T or G or C, D Represents the base G or A or T, K represents the base G or T.

[0053] Table 1. Multiplex PCR primer sequences for detection of RNA viruses

[0054]

[0055] Note: F means forward primer; R means reverse primer

[0056] 2. Probe design:

[0057] The present invention designs a total of 12 probes for 7 kinds of inse...

Embodiment 2

[0091] According to the method of Example 1, 10 positive clinical samples confirmed to be dengue virus type 1 infection by antigen method and PCR method were detected, and the positive detection rate results are shown in Table 5.

[0092] Table 5. The positive detection rate of dengue virus type 1 by optimized primers

[0093]

[0094] It can be seen from Table 5 that after the degenerate base design of the primers, the positive detection rate is significantly improved, and positive samples can be detected to the greatest extent.

Embodiment 3

[0096] A pregnant woman with fever and muscle soreness symptoms is detected to be dengue virus positive with the dengue antigen method, but it is not known to be the dengue virus of that type, and it is determined to be dengue type 2 virus according to the method of embodiment 1 (see figure 1 ), the result was the same as the antigen method and PCR method.

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