CN106755600A - A kind of PCR kit for fluorescence quantitative for detecting Zaire type Ebola virus - Google Patents

Abstract

The invention relates to a fluorescent quantitative PCR kit for detecting Zaire-type Ebola virus. The kit includes primers, TaqMan fluorescent probes and positive standards, and is characterized in that: (1) the primers are amplified A pair of primers for highly conserved fragments of the coding gene of the Zaire type Ebola virus envelope glycoprotein, wherein the upstream primer sequence is as shown in SEQ NO.1, and the downstream primer sequence is as shown in SEQ NO.2; (2) The sequence of the TaqMan fluorescent probe is shown in SEQ NO.3; (3) the positive standard is a recombinant plasmid containing the DNA fragment of the sequence shown in SEQ NO.4. The kit of the invention has the advantages of good specificity and high sensitivity.

Description

A fluorescent quantitative PCR kit for detecting Zaire-type Ebola virus

technical field

The invention relates to the field of biochemistry, in particular to a detection reagent for a determination or detection method comprising nucleic acid, enzyme or microorganism.

Background technique

Ebola virus is a filoviridae virus that can cause severe zoonotic infectious diseases. It can cause Ebola hemorrhagic fever. It has the characteristics of high pathogenicity and high fatality rate. One of the most serious viruses for humans, biosafety level 4 (BSL-4).

At present, the routine detection methods of Zaire Ebola virus in my country include serological reaction, nucleic acid hybridization, antigen-antibody ELISA and other technologies, but these tests or methods are cumbersome to operate and take a long time (about 2 days or more). Existing Zaire-type Ebola virus detection technologies are still few, and most of them are unreliable, with low sensitivity and poor specificity. The nucleic acid detection method promoted in the past was found to have serious false negatives in the 2014 West African epidemic, indicating that the detection primers and probes for Zaire Ebola virus urgently need to be updated. This patent aims to establish a TaqMan probe real-time fluorescent quantitative PCR method for detecting Zaire-type Ebola virus, and update and promote the past methods.

Contents of the invention

The technical problem to be solved by the present invention is to provide a fluorescent quantitative PCR kit for detecting Zaire-type Ebola virus, which has the advantages of good specificity and high sensitivity.

The technical scheme that the present invention solves the problems of the technologies described above is:

A fluorescent quantitative PCR kit for detecting Zaire type Ebola virus, the kit includes primers, TaqMan fluorescent probes and positive standards, characterized in that:

(1) The primers are a pair of primers for amplifying a highly conserved fragment of the gene encoding the Zaire-type Ebola virus envelope glycoprotein, wherein,

The upstream primer sequence is: 5'-GAACCACATGATTGGACCAAGA-3' (SEQ NO.1),

The downstream primer sequence is: 5'-TAACTCCAATACCTGCCGGTATC-3' (SEQ NO.2);

(2) the TaqMan fluorescent probe sequence is:

5'FAM-TTGTTGATAAAACCCTTCCGGACCAGG-BHQ 3'(SEQ NO.3);

(3) The positive standard product is a recombinant plasmid containing the DNA fragment of the following sequence:

TGGGACCGGACTGCTGTATCGAACCACATGATTGGACCAAGAACATAACAGACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGACCAGGGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGG (SEQ NO. 4).

In the above-mentioned scheme, described positive standard substance is made by following method:

1) According to the coding gene sequence of the Zaire Ebola virus envelope glycoprotein published on the network database GenBank, use the Clustal_X software to conduct sequence homology analysis, and then compare and analyze its specificity with the blastn network tool to find the The amino acid sequence of the highly conserved region in the encoding gene, and then synthesize the DNA fragment of the amino acid sequence according to conventional methods;

2) inserting the DNA fragment synthesized in step (1) into the plasmid vector pUC57 (Amp ⁺ ) to construct a recombinant plasmid;

3) Take 5 μl of the recombinant plasmid obtained in step (1) and mix it with 100 μl of DH5α competent cells, and put it in an ice bath for 20 minutes; after heat shocking in a water bath at 42°C for 70-90 seconds, quickly put it in an ice bath for 5 minutes; add 500 μl of LB meat Soup culture medium, mixed, cultured on a shaker at 37°C 200rpm for 1 hour to obtain a bacterial solution; take 100 μl of bacterial solution in a mixture containing ampicillin at a concentration of 100 μg/μl, X-gal at a concentration of 20 μg/μl and X-gal at a concentration of 200 μg/μl Spread on the LB medium plate of IPTG and cultivate for 18 hours; then select the white strains on the medium for expansion culture through blue-white screening;

4) Extract the white bacterial strain after expansion according to the requirements of the rapid plasmid extraction kit provided by Omega, and then dilute it with deionized water to 10 ⁶ copies/μl to obtain a positive standard.

The using method of the test kit of the present invention is as follows:

(1) The reaction system is as shown in Table 1:

Table 1 PCR reaction system of the kit of the present invention

(3) Quantitative standard curve preparation: Dilute the quantitative positive template into 7 concentration gradients of 10 ⁹ -10 ³ copies/μl according to the 10-fold dilution method, and perform amplification according to the reaction system and reaction procedure described in steps (1) and (2). increase;

(4) Sample detection: After the positive standard, negative control and sample RNA to be tested are prepared according to the above reaction system and the reaction procedure is amplified, the corresponding Ct value of each sample is obtained from the supporting software of the instrument;

(5) Judgment of results: Ct value ≤ 35.0 is positive, no value or greater than 35.0 is negative.

The kit of the present invention has higher specificity compared with kits of commonly used serological methods; compared with the past Ebola virus fluorescence quantitative PCR detection kit, it can detect Zaire type Ebola more rapidly and accurately Bora virus, the detection time is 45min, and the minimum detection limit reaches 10copies/μl. It has good specificity and can be used for clinical diagnosis, epidemic prevention and monitoring of infectious diseases, entry-exit inspection and quarantine and Zaire Ebola virus in related fields Qualitative and quantitative rapid real-time nucleic acid detection.

Description of drawings

Fig. 1 is a position map of the amino acid sequence SEQ NO.4 of the highly conserved region in the coding gene in the Zaire-type Ebola virus genome.

Fig. 2 is a standard curve drawn after PCR amplification using the kit of the present invention.

Fig. 3 is a graph showing the sensitivity test results of the kit of the present invention in Example 2, in which the starting template concentrations are 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , and 10 ¹ from left to right in the figure. For the amplification curve of copies/μl, the bottom curve (with a small Ct value) near the abscissa is the negative control.

Fig. 4 is the curve diagram of the specificity determination result of the kit of the present invention in Example 2, in which the corresponding starting templates from left to right are respectively the amplification curves of the positive standard and the DH5α engineering bacteria containing the recombinant plasmid, without the Ct value The template of the curve is the amplification curve of type I dengue virus RNA, Zika virus RNA, negative control.

detailed description

The beneficial effects of the present invention will be described below in conjunction with examples.

Embodiment 1: (preparation kit)

1. The composition of the fluorescent quantitative PCR kit for detection of Zaire Ebola virus:

Upstream primer: 5'-GAACCACATGATTGGACCAAGA-3', 10μM;

Downstream primer: 5'-TAACTCCAATACCTGCCGGTATC-3', 10μM;

Fluorescent probe: 5'FAM-TTGTTGATAAAACCCTTCCGGACCAGG-BHQ 3', 10μM;

Positive standard: pUC57(Amp ⁺ ) recombinant plasmid containing DNA fragment SEQ NO.4, the concentration is 10 ⁶ copies/μl.

Polymerase mixture: containing 5U/μl Taq DNA polymerase and 200U/μl reverse transcriptase, purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.;

PCR reaction buffer (5×): purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.;

The above nucleic acids were all synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

2. The preparation method of the quantitative positive template is:

(1) According to the coding gene sequence of the Zaire Ebola virus envelope glycoprotein published on the network database GenBank, use the Clustal_X software to conduct sequence homology analysis, and then compare and analyze its specificity with the blastn network tool to find A highly conserved region in the coding gene, and then submit the sequence information to have the DNA fragment of SEQ NO.4 synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

(2) Insert the fragment into the plasmid vector pUC57 (Amp ⁺ ) to construct a recombinant plasmid, the specific method is:

a. Ligation and transformation of DNA fragment SEQ NO.4 and plasmid vector pUC57 (Amp ⁺ ):

1) Add to a microcentrifuge tube:

pUC57(Amp ⁺ ) 1μl

DNA fragment (SEQ NO.4) 4μl

dH ₂ O 4 μl;

2) Add 1 μl of T4DNA Ligase;

3) React overnight at 16°C and connect;

4) Take 100 μl of competent cells DH5α in a centrifuge tube and pre-cool in ice for 20 minutes;

5) Add 5 μl of the ligation product;

6) After mixing gently, pre-cool in ice for 30 minutes;

7) 42℃70～90S thermal shock treatment;

8) Quickly move to ice for cooling for 5 minutes;

9) Add 500 μl of LB liquid medium at 37°C;

10) 37°C, 200r/min shaking culture for 1h;

11) Take 100 μl of medium and spread it on an LB medium plate containing 100 μg/μl ampicillin, 20 μg/μl X-gal, and 200 μg/μl IPTG;

12) Cultivate at 37°C for 18 hours;

13) Take 5 colonies and put them into 4 ml of LB culture medium containing 100 μg/μl ampicillin and amplify them by shaking for 18 hours.

b. Recombinant plasmid extraction:

1) Take 50ml of the amplified bacteria solution and centrifuge at 5000g for 10min;

2) Discard the supernatant and add 250 μl SolutionⅠ/R NaseA to suspend the cells;

3) Add 250 μl Solution II, mix gently, and turn it upside down 4 to 6 times;

4) Add 350 μl SolutionⅢ, mix gently, and turn it upside down until white flocs are formed;

5) Centrifuge at 13,000g for 10min;

6) Carefully add the clear supernatant to the clean HiBind ^TM DNA Miricolumn, and centrifuge the Miricolumn at 10,000g for 1 min;

7) Remove the liquid, add 500μl bufferHB to wash the tube, and centrifuge at 10,000g for 1min;

8) Discard the liquid, add 700μl DNA wash buffer, centrifuge at 10,000g for 1min;

9) Repeat step 8);

10) Then centrifuge the empty tube at 13,000g for 3min;

11) Put the empty tube into a clean 1.5ml centrifuge tube, add 60μl of deionized water, and centrifuge at 13,000g for 2min.

3. Identification: The obtained recombinant plasmid was subjected to ordinary PCR with designed primers F and R, and electrophoresed on 2.0% agarose gel to obtain a band size of 145 bp. The samples were diluted to 10 ⁶ copies/μl after measuring the concentration.

The length of the sequence shown in the above-mentioned SEQ NO.4 is 169 bp, which is between 1949 and 2117 bp of the genome sequence of the Zaire type Ebola virus (see FIG. 1 ).

The kits used in the following examples are the kits described in Example 1.

Embodiment 2: (in vitro detection experiment)

1. Preparation of the template:

a. Reagents: RNA extraction kit, purchased from Axygen.

b. Cultivation and preparation of standard strains:

Positive standard: 10 ⁶ copies/μl recombinant plasmid;

Samples to be tested: DH5ɑ engineering bacteria containing recombinant plasmids are used as positive samples, animal cell culture medium of type I dengue virus and Zika virus and DH5ɑ strains without recombinant plasmids are used as mock samples, and the bacteria do not need to extract RNA and use the whole genome as a template ;

Negative control: deionized water.

c. RNA extraction: take 200 μl of animal cell culture medium containing type I dengue virus and Zika virus, and extract viral RNA with an RNA extraction kit, with a final volume of 30 μl (for detailed steps, please refer to the kit instruction manual). The extracted RNA template was stored at -80°C for later use.

2. Fluorescent quantitative PCR reaction:

1) The reaction system is as shown in Table 2:

Table 2 PCR reaction system of the kit of this embodiment

2) Method: each batch of reactions is provided with a positive control and a negative control (replaced by deionized water), and the reaction procedure is:

Instrument: Roche real-time fluorescence quantitative PCR instrument LightCycler 96, Roche Company (Switzerland).

3) Quantitative standard curve preparation:

Dilute the recombinant plasmid template by 10 times to a total of 7 concentration gradients of 10 ⁹ to 10 ³ copies/μl, and perform amplification according to the above reaction system and reaction procedure. After the reaction, the computer automatically draws a standard curve, as shown in Figure 2 . The linearity of the starting template was good at 10 ⁹ -10 ³ copies/μl, and the standard curve was obtained by software analysis: Y=-3.7514X+43.31, R ² =0.999.

4) Sensitivity determination:

Take the positive standard product and dilute it by 10 times to 10 ⁶ to 10 ¹ copies/μl, a total of 6 concentration gradients, amplify according to the above reaction system and reaction procedure, and analyze with fluorescent quantitative PCR to determine the lower limit of detection, as shown in the figure 3. The Ct value of the starting template was 34.44 when the starting template was 10 ¹ copies/μl, which indicated that the detection limit of the detection method was 10 ¹ copies/μl.

5) Specificity determination

Positive standard samples were taken as positive controls, DH5α engineering bacteria containing recombinant plasmids were used as positive samples, RNA of type I dengue virus, Zika virus and DH5α strains without recombinant plasmids were used as mock samples, and deionized water was used as negative control. The above reaction system and reaction program were amplified to test the specificity of the reaction, and the results are shown in FIG. 4 . The fluorescent quantitative PCR results of the positive control and positive samples showed regular amplification curves, and the Ct was less than 35, which was positive; the detection results of dengue virus type I, Zika virus RNA and DH5ɑ strain without recombinant plasmid as mock samples were all negative. Ct value was negative. It was completely consistent with the expected results, and there was no non-specific amplification.

SEQUENCE LISTING

<110> Southern Medical University

<120> A Fluorescent Quantitative PCR Kit for Detecting Zaire Ebola Virus

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 22

<212>DNA

<213> Artificial sequence

<400> 1

gaaccacatg attggaccaa ga 22

<210> 2

<211> 23

<212>DNA

<213> Artificial sequence

<400> 2

taactccaat acctgccggt atc 23

<210> 3

<211> 27

<212>DNA

<213> Artificial sequence

<400> 3

ttgttgataa aacccttccg gaccagg 27

<210> 4

<211> 169

<212>DNA

<213> Artificial sequence

<400> 4

tgggaccgga ctgctgtatc gaaccacatg attggaccaa gaacataaca gacaaaattg 60

atcagattat tcatgatttt gttgataaaa cccttccgga ccaggggggac aatgacaatt 120

ggtggacagg atggagacaa tggataccgg caggtattgg agttacagg 169

Claims (2)

1. A fluorescent quantitative PCR kit for detecting Zaire type Ebola virus, the kit includes primers, TaqMan fluorescent probes and positive standards, characterized in that: (1) The primers are a pair of primers for amplifying a highly conserved fragment of the gene encoding the Zaire-type Ebola virus envelope glycoprotein, wherein, The upstream primer sequence is: 5'-GAACCACATGATTGGACCAAGA-3' (SEQ NO.1), The downstream primer sequence is: 5'-TAACTCCAATACCTGCCGGTATC-3' (SEQ NO.2); (2) the TaqMan fluorescent probe sequence is: 5'FAM-TTGTTGATAAAACCCTTCCGGACCAGG-BHQ 3'(SEQ NO.3); (3) The positive standard product is a recombinant plasmid containing the DNA fragment of the following sequence: TGGGACCGGACTGCTGTATCGAACCACATGATTGGACCAAGAACATAACAGACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGACCAGGGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGG (SEQ NO. 4). 2. a kind of fluorescent quantitative PCR kit that detects Zaire type Ebola virus according to claim 1, is characterized in that, described positive standard item is made by following method: 1) According to the coding gene sequence of the Zaire Ebola virus envelope glycoprotein published on the network database GenBank, use the Clustal_X software to conduct sequence homology analysis, and then compare and analyze its specificity with the blastn network tool to find the The amino acid sequence of the highly conserved region in the encoding gene, and then synthesize the DNA fragment of the amino acid sequence according to conventional methods; 2) inserting the DNA fragment synthesized in step (1) into the plasmid vector pUC57 (Amp ⁺ ) to construct a recombinant plasmid; 3) Take 5 μl of the recombinant plasmid obtained in step (1) and mix it with 100 μl of DH5α competent cells, and put it in an ice bath for 20 minutes; after heat shocking in a water bath at 42°C for 70-90 seconds, quickly put it in an ice bath for 5 minutes; add 500 μl of LB meat Soup culture medium, mixed, cultured on a shaker at 37°C 200rpm for 1 hour to obtain a bacterial solution; take 100 μl of bacterial solution in a mixture containing ampicillin at a concentration of 100 μg/μl, X-gal at a concentration of 20 μg/μl and X-gal at a concentration of 200 μg/μl Spread on the LB medium plate of IPTG and cultivate for 18 hours; then select the white strains on the medium for expansion culture through blue-white screening; 4) Take the white bacterial strain after expansion, extract according to the requirements of the rapid plasmid extraction kit provided by Omega, and then dilute with deionized water to 10 ⁶ copies/μl to obtain the positive standard.