CN106636461A - Detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV and STLV - Google Patents

Abstract

The invention discloses a detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV and STLV. The detection primer set includes specific sequence primers of three different viruses, SIV, SRV, and STLV, and has a specific Tag sequence. The detection method is to extract the total virus DNA in the blood sample, and simultaneously amplify the target fragments of the three viruses through multiplex PCR, and then hybridize the amplified products with fluorescently encoded microspheres and streptavidin-phycoerythrin solution, Read the MFI value through the detection instrument to determine whether it contains these three types of viruses. The invention can simultaneously detect and distinguish three different types of viruses: SIV, SRV and STLV. The invention has the advantages of high specificity, high sensitivity, fast and high efficiency, low cost, accurate and easy-to-read results, and is very suitable for popularization and application in various detection units.

Description

A detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV, and STLV

technical field

The invention belongs to the technical field of molecular biology and relates to a detection method for pathogens related to experimental animals, in particular to a detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV and STLV.

Background technique

Non-human primates are closely related to humans in evolution, and have the characteristics of wide natural distribution, large numbers, and easy breeding. They are widely used in the research of reproductive physiology, drug toxicology, biological products and human disease models. my country is rich in non-human primate resources, which are mainly concentrated in southern regions such as Guangdong, Guangxi, Yunnan, Sichuan, and Hainan. There are currently more than 50 breeding companies with a population of more than 300,000 monkeys, which are used for scientific research every year. There are more than 30,000 pieces, and more than 20,000 pieces are exported every year. With the increase of experimental monkeys and breeding enterprises, the pathogens carried by monkeys have gradually attracted people's attention, which makes the chances of human beings infected with monkeys significantly increased during the process of contact with monkeys, especially those that are currently difficult to cure such as AIDS. The emergence of other diseases has increased the risk of occupational personnel. In addition, the pathogens carried by experimental monkeys also seriously affect the accuracy and authenticity of scientific research results and damage the economic benefits of enterprises. At present, the three most commonly carried viruses in experimental monkeys are simian D retrovirus (SRV), simian immunodeficiency virus (SIV) and simian T lymphotropic virus (STLV).

In the existing experimental animal quality testing standards, the detection methods for the above three pathogens still use ELISA or IFA detection methods, which have poor specificity, low sensitivity, and high false positives, and must also be combined with WB detection specificity Antibodies are the only way to make a definitive diagnosis. The high false positive rate of ELISA and IFA and the complicated operating procedures of WB method limit their application to a certain extent. Therefore, it is necessary to establish a convenient, fast, sensitive and specific detection method, whether it is to protect the health of the entire breeding monkey group or the personal safety of the breeding personnel and scientific researchers.

Contents of the invention

One object of the present invention is to provide a detection primer set that can simultaneously detect and distinguish SIV, SRV, and STLV.

Another object of the present invention is to provide a method for simultaneously detecting and distinguishing SIV, SRV, and STLV.

The technical scheme that the present invention takes is:

A detection primer set capable of simultaneously detecting and distinguishing SIV, SRV, and STLV, including three pairs of primers, the nucleotide sequence of which is as follows:

SIV primers:

SIV-F: 5'-ACGACCCGGCGGAAAGAAAAAGT-3' (SEQ ID NO: 1);

SIV-B: 5'-TGCACCAGATGACGCAGACAGT-3' (SEQ ID NO: 2);

SRV primers:

SRV-F: 5'-AYGGGGCTACTGCYCCATA-3' (SEQ ID NO: 3);

SRV-B: 5'-GCCATTACCKGCYTGTTGATT-3' (SEQ ID NO: 4);

STLV primers:

STLV-F: 5'-GTGCCAATCATGGACCTGCC-3' (SEQ ID NO: 5);

STLV-B: 5'-TCCTGGAGCGTCGATTAGAAGG-3' (SEQ ID NO: 6).

Preferably, the 5' ends of the primer sequences of primers SIV-F, SRV-F, and STLV-F are added with a Tag sequence through a spacer sequence.

Preferably, the Tag sequences of primers SIV-F, SRV-F, and STLV-F primers are respectively:

The Tag sequence of SIV-F is: 5'-ATCTCAATTACAATAACACACAAA-3' (SEQ ID NO: 7);

The Tag sequence of SRV-F is: 5'-TACTTCTTTTACTACAATTTACAAC-3' (SEQ ID NO: 8);

The Tag sequence of STLV-F is: 5'-CTTAAACTCTACTTACTTCTAATT-3' (SEQ ID NO: 9).

Preferably, the 5' end of the primer sequence of primer SIV-R, SRV-R, STLV-R is added with a biotin label.

A method capable of simultaneously detecting and distinguishing SIV, SRV, and STLV, comprising the steps of:

1) Extract RNA and synthesize cDNA from the sample to be tested;

2) PCR amplification reaction;

3) PCR products are hybridized with fluorescently encoded microspheres.

4) Signal detection and result interpretation.

Preferably, the PCR amplification reaction program is pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and 33 cycles; extension at 72°C for 5 min.

Preferably, the hybridization system between the PCR product and the fluorescently encoded microsphere solution is: 20 μl of the fluorescently encoded microsphere solution, 75 μl of the streptavidin-phycoerythrin solution, and 5 μl of the PCR product.

Preferably, the fluorescently encoded microspheres are respectively marked with a sequence reversely complementary to the aforementioned Tag sequence.

Preferably, the procedure for hybridizing the PCR product with the fluorescently encoded microspheres is: hybridize at 45° C. for 35 minutes.

The beneficial effects of the present invention are:

The present invention has beneficial effects such as high specificity, high sensitivity, fast and efficient, low cost, accurate and easy-to-read results:

1) High specificity: According to the highly conserved sequences of SIV, SRV and STLV, the present invention designs three sets of specific primers respectively. The Tm values of the three sets of primers are close, and high annealing temperature can be accepted for PCR reaction, which ensures that Multiple PCR reactions and high specificity;

2) High sensitivity: Since the detection result is judged by the fluorescent signal reading method, the detection method is 1 to 2 orders of magnitude higher than the traditional PCR detection method.

3) Fast and efficient: Only one sample can be extracted to detect and distinguish three viruses at the same time, which greatly shortens the detection time and sample size;

3) Low cost: Since only one reagent is needed to detect multiple indicators of one sample, it breaks the limitation of the traditional method that one reagent can detect one indicator of one sample, realizes real high-throughput detection, and greatly reduces the detection cost. the cost of reagents;

4) The result is accurate and easy to read: the detection fluorescence signal is read by Luminex 200 instrument without electrophoresis, the detection result is easy to judge, and the accuracy is high. This detection method meets the needs of modern detection.

Description of drawings

Fig. 1 is the detection result figure of embodiment 3 specificity;

Fig. 2 is the sensitivity detection result figure of each virus plasmid of embodiment 4.

detailed description

The present invention will be further described below in conjunction with embodiment, but is not limited thereto.

Embodiment 1 can simultaneously detect and distinguish the design of the detection primer set of SIV, SRV, STLV

The main core protein gag gene partial sequence of simian retrovirus (SIV) (GenBank accession number is M74931.1), part of the conserved gene sequence in the p27 protein of simian retrovirus (SRV1, SRV2, SRV3) (GenBank accession number Numbers are M11841.1, AF126467.1, M12349), and the env gene partial sequence of monkey T lymphotropic virus type Ⅰ is mainly the target gene. Primer Premier 6 is used to design specific primers, and the designed upstream primers are respectively A Tag sequence was added to the 5' end, and Spacer C18 was used to separate the Tag sequence from the primer sequence, and a biotin label was added to the 5' end of the downstream primer. The sequences of the detection primers used for PCR amplification are shown in Table 1.

Table 1. SIV, SRV, STLV detection primer set

Example 2 Establishment of a method for simultaneously detecting and distinguishing SIV, SRV, and STLV.

(1) Extraction of total viral RNA: The total viral RNA in the SIV and SRV virus culture medium was extracted by using the total viral RNA extraction kit of Meiji Biotechnology Co., Ltd., and reverse-transcribed into cDNA by using the reverse transcription kit.

(2) PCR amplification reaction:

The 20 μl reaction system contains: SIVfd 0.2 μM, SIVbk 0.2 μM, SRVfd 0.2 μM, SRVbk 0.2 μM, STLV1fd 0.2 μM, STLVbk 0.2 μM, multiplex PCR Assay Kit reaction solution 10.0 μl, PCR EnzymeMix 0.1 μl, cDNA to be tested 2.0 μl, Make up to 20 μl with DEPC water; set positive control and negative control; mix the prepared PCR tube and centrifuge, and perform PCR reaction according to the following procedure: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30s, 58°C for 30s, 72°C Extend at ℃ for 30s, cycle 33 times; extend at 72℃ for 5min.

(3) Hybridization of PCR products with fluorescently encoded microspheres.

A. Preparation of fluorescently encoded microsphere solution: fluorescent microspheres coated with reverse complementary Tag sequences to the designed primers (three kinds of microspheres were purchased from Luminex, the concentration was 2500/μl, specific SIV, SRV, STLV Corresponding fluorescent coded microsphere numbers are MTAG-67, MTAG-15, MTAG-56) were diluted with 1.1×Hybrdization Buffer to contain 50 fluorescent coded microspheres per microliter solution.

B. Prepare streptavidin-phycoerythrin solution: Dilute 1mg/ml SAPE solution to 10ug/ml with 1×HybrdizationBuffer.

C. Prepare hybrid system:

Reagent volume Fluorescence-encoded microsphere solution 20μl Streptavidin-phycoerythrin solution 75μl PCR product 5μl total capacity 100μl

D. Hybridization procedure: hybridization at 45°C for 35 minutes

(4) Signal detection and result interpretation.

The hybridization products were detected and analyzed by Luminex 200 instrument, the MFI value of each fluorescent microsphere in each sample was read and the average MFI value was calculated as the basis for judgment. According to the instrument software settings, select the negative control as a reference, when the MFI value of the sample is greater than 1000, it can be judged as positive.

Embodiment 3 specificity experiment

The mixed cDNA templates of SIV, SRV, and STLV were simultaneously detected by the method in Example 2 to verify the specificity.

The test results showed that the three viral primers had no cross-reaction with each other (see Figure 1).

Embodiment 4 Sensitivity experiment

Using the cDNA of each virus as a template, the target fragments were respectively amplified with SIV-F/SIV-R, SRV-F/SRV-R, and STLV-F/STLV-R primer sets and cloned into the pMD19-T vector , to construct a specific plasmid, and then carry out a 10-fold gradient dilution, and use the operation method of Example 2 to detect the sensitivity of each gradient plasmid DNA after dilution.

The test results showed that: for SIV, the detection sensitivity of the SIVfd/SIVbk primer set to the plasmid was 10fg/μl; for SRV1, SRV2, and SRV3, the detection sensitivity of the SRVfd/SRVbk primer set to the plasmid was 100fg/μl, 10fg/μl, and 10fg, respectively /μl, for STLV, the detection sensitivity of the STLVfd/STLVbk primer set to the plasmid is 1fg/μl (see Figure 2).

The comparison of different primer sets detection effect of embodiment 5

Using the method of Example 2, other detection primer sets were used for sensitivity detection of plasmids of various concentrations according to the method of Example 4. The sequences and detection results of other detection primer sets are shown in Table 2 and Table 3.

Table 2 Other Detection Primer Set Sequences

Table 3 Comparison of detection effects of different detection primer groups

Note: Detection primer set A is from the primer set of the present invention.

The test result shows: the detection primer set of the present invention has higher detection sensitivity than other primer sets, and the interference between the primers is smaller; there is no false positive in primer set B, but the detection sensitivity has decreased; the detection primer set C has higher detection sensitivity. The negative control test was positive, indicating interference with the detection primer set.

The invention has the advantages of high specificity, high sensitivity, fast and high efficiency, low cost, accurate and easy-to-read results, etc., and is suitable for popularization and application in the detection process of experimental animal pathogens by detection units at all levels.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Guangdong Laboratory Animal Monitoring Institute

<120> A detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV, and STLV

<130>

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Claims (9)

1. a kind of while detect and distinguish the detection primer group of SIV, SRV, STLV, including three pairs of primers, its nucleotide sequence is such as Shown in lower：

SIV primers：

SIV-F：5’-ACGACCCGGCGGAAAGAAAAAGT-3’（SEQ ID NO:1）；

SIV-B：5’-TGCACCAGATGACGCAGACAGT-3’（SEQ ID NO:2）；

SRV primers：

SRV-F：5’-AYGGGGCTACTGCYCCATA-3’（SEQ ID NO:3）；

SRV-B：5’-GCCATTACCKGCYTGTTGATT-3’（SEQ ID NO:4）；

STLV primers：

STLV-F：5’-GTGCCAATCATGGACCTGCC-3’（SEQ ID NO:5）；

STLV-B：5’-TCCTGGAGCGTCGATTAGAAGG-3’（SEQ ID NO:6）.

2. detection primer group according to claim 1, it is characterised in that：Primer SIV-F, SRV-F, STLV-F primer sequence 5 ' ends of row are added with Tag sequences by spacer sequence.

3. detection primer group according to claim 2, it is characterised in that：The Tag sequences are respectively：

The Tag sequences of SIV-F are：5’-ATCTCAATTACAATAACACACAAA-3’（SEQ ID NO:7）；

The Tag sequences of SRV-F are：5’-TACTTCTTTACTACAATTTACAAC-3’（SEQ ID NO:8）；

The Tag sequences of STLV-F are：5’-CTTAAACTCTACTTACTTCTAATT-3’（SEQ ID NO:9）.

4. detection primer group according to claim 1, it is characterised in that：Primer SIV-R, SRV-R, STLV-R primer sequence 5 ' ends of row are added with biotin labeling.

5. a kind of method that can simultaneously detect SIV, SRV, STLV, comprises the steps：

1）Extract measuring samples RNA and synthesis cDNA；

2）Pcr amplification reaction；

3）PCR primer hybridizes with fluorescence-encoded micro-beads；

4）Signal detection and result interpretation.

6. detection method according to claim 5, it is characterised in that：Pcr amplification reaction program is 94 DEG C of denaturations 5min； 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of 30 s of extension, circulate 33 times；72 DEG C of extension 5min.

7. detection method according to claim 5, it is characterised in that：The hybridization of PCR primer and fluorescence-encoded micro-beads solution System is：The μ l of fluorescence-encoded micro-beads solution 20, the μ l of SA-PE solution 75, the μ l of PCR primer 5.

8. detection method according to claim 5, it is characterised in that：The fluorescence-encoded micro-beads are marked with and claim The sequence of the Tag sequence reverse complementals described in 2.

9. detection method according to claim 5, it is characterised in that：The program that PCR primer hybridizes with fluorescence-encoded micro-beads For 45 DEG C of hybridization 35min.