CN101333568B - Quantitative determination process for enterovirus in environment water body - Google Patents

Abstract

The invention discloses a method for quantitative detection of enteroviruses in environmental water bodies. According to the homology of the conserved sequence of the 5' non-coding region of enterovirus RNA, the method is designed on the basis of existing primers EV1 and EV2. For the common primer EVN of enterovirus, a series of gradiently diluted recombinant plasmids were prepared as standard products, and a real-time fluorescent quantitative PCR reaction system using the universal primer EVN of enterovirus was established to quantitatively detect the content of enterovirus in environmental water. The method is quantitatively accurate, greatly improves the sensitivity of the existing qualitative RT-PCR detection technology for enteroviruses in environmental water, has a wide linear range, high precision, and good specificity, which is different from other common pathogenic microorganisms in the environment. Cross reaction.

Description

A quantitative detection method for enteroviruses in environmental water

technical field

The invention relates to a quantitative detection method for enteroviruses in environmental water bodies. The method uses a real-time fluorescent quantitative RT-PCR method to detect enteroviruses, and is suitable for quantitative determination of various viruses of the genus Enterovirus in environmental water bodies.

Background technique

Reverse transcription-polymerase chain reaction (RT-PCR) technology uses RNA as a template to reverse transcribe the first strand of complementary DNA, and then use this strand as a template to perform PCR reaction, so that a very small amount of RNA can be synthesized within a few hours The internal specificity is amplified by millions of times. Real-time fluorescent quantitative RT-PCR technology adds fluorescent groups to the reaction system, uses fluorescent signals to monitor the entire PCR process in real time, and finally uses the standard curve to quantitatively analyze the concentration of unknown templates. Most of the existing PCR detection methods for enteroviruses in environmental waters are based on qualitative RT-PCR detection technology, which has the disadvantages of inaccurate quantification, insufficient sensitivity, and greater influence by impurities. Although real-time fluorescent quantitative RT-PCR technology has been applied to the detection of microorganisms in the medical field, it is not suitable for the needs of environmental water bodies. Therefore, there is currently no real-time fluorescent quantitative RT-PCR method suitable for the detection of enteroviruses in environmental water bodies.

Contents of the invention

In view of the defects or deficiencies in the above-mentioned prior art, the purpose of the present invention is to provide a quantitative detection method for enteroviruses in environmental water, so as to solve the problem of quantitative detection and improve the sensitivity and specificity of detection.

In order to realize above-mentioned task, the present invention takes following technical solution:

A quantitative detection method for enteroviruses in environmental water, characterized in that it comprises the following steps:

Step 1, enterovirus universal primer EVN design:

According to the homology of the conserved sequence of the enterovirus RNA 5' non-coding region, on the basis of the existing primers EV1 and EV2, a pair of enterovirus universal primers EVN is designed, and the enterovirus universal primer EVN includes the upstream Primer EVN1 and downstream primer EVN2, wherein the nucleotide sequence of the upstream primer EVN1 is: 5'-ACTTCGAGAAGCCTAGTACC-3'; the sequence of the downstream primer EVN2 is: 5'-TAGGATTAGCCGCATTCAG-3';

Step 2, preparation of standard substance:

Inoculate poliovirus into Vero cells. When the cell virus reaches 3+~4+, collect the diseased cells, extract RNA from the sample, use EV2 as a primer, add AMV reverse transcriptase, and perform reverse transcription according to conventional methods. Store the reverse transcription product at -20°C;

The reverse transcription product was used as a template, and EV1 and EV2 were used as primers to carry out PCR amplification, and the obtained target product fragment was recovered and purified, ligated with pMD18-T vector and transformed into Escherichia coli. The positive colonies were screened out on a plate, cultured in LB liquid medium containing Amp, the plasmid was extracted for sequence determination, quantified by a UV spectrophotometer, and stored at -20°C after gradient dilution as a standard for fluorescent real-time quantitative PCR;

Using recombinant plasmids with ten concentration gradients ranging from 2.31GEC/μL to 2.31×10 ⁹ GEC/μL as templates, real-time fluorescent quantitative PCR was carried out according to the measurement procedure. Samples of each concentration were measured 5 times, and the average value was used for regression to obtain a standard curve. equation:

C _T ＝-3.6896logX ₀ +42.987(R ² ＝0.997) (Formula 1)

In the formula: X ₀ ——Initial template amount (GEC)

Enterovirus content in water samples: 40X ₀ (GEC) (Formula 2)

Step three, quantitative detection:

1) Sample collection: Take water samples at 0.3m below the water surface, and store them in a 4°C ice box after collection;

2) Preliminary concentration of virus: the mixed cellulose ester filter membrane is packed into a vacuum filter, the water sample containing enterovirus is filtered, the membrane is eluted with beef extract-glycine buffer as the eluent, and the eluate is collected;

3) Secondary concentration: add PEG and NaCl to the eluent, let it stand overnight at 4°C, stir and mix well, let stand at 4°C to precipitate overnight, centrifuge, and collect the precipitate;

4) RNA extraction: use the RNA extraction kit to extract viral RNA from the precipitate;

5) Reverse transcription: mix 13 μL RNA and 1 μL 25 μmol/L EV2 primer, add AMV reverse transcriptase, bathe in 70°C water for 10 minutes, ice bath for 10 minutes, and after centrifugation, add 2 μL 10×RTbuffer, 2 μL dNTP mixture (total 40 mmol/L L), 10U RNase inhibitor and 5U AMV enzyme, in a water bath at 43°C for 1 hour, and in a water bath at 70°C for 10 minutes, centrifuged to obtain the reverse transcription product as a PCR template;

6) Real-time fluorescent quantitative RT-PCR detection: Take 12.5 μL of 2×SYBR Mix, 0.5 μL each of 10 μmol/L upstream primer EVN1 and 10 μmol/L downstream primer EVN2, 2 μL 25 mmol/L MgCl ₂ , Taq enzyme 2U, add 2 μL of template, Make up to 25 μL with sterile ultrapure water, use sterile ultrapure water as a template as a negative control, and perform amplification and result analysis on a real-time quantitative PCR instrument;

The reaction conditions are:

① Pre-denaturation at 94°C for 4 minutes;

② Amplification cycle 40 times: 94°C, 30s; 55°C, 30s; 72°C, 30s;

③Collect fluorescence signal at 85°C, perform melting curve analysis after the cycle ends, heat up from 65°C to 95°C at a rate of 0.2°C/s, and continuously detect fluorescence during the whole process;

7) Calculation: According to the C _T value obtained from the detection, substitute into the standard curve equation 1 to calculate the initial template amount of enterovirus, and then substitute into equation 2 to calculate the content of enterovirus in the sample.

The detection method of the present invention is quantitatively accurate, greatly improves the sensitivity of the existing qualitative RT-PCR detection technology for enteroviruses in environmental water bodies, has a wide linear range, high precision, and good specificity. Pathogenic microorganisms do not cross-react.

Description of drawings

Fig. 1 is the recombinant plasmid amplification curve of gradient template amount;

Figure 2 is the standard curve of enterovirus real-time fluorescent quantitative PCR.

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

Detailed ways

According to the technical scheme of the present invention, firstly, according to the homology of the conserved sequence of the enterovirus RNA 5' non-coding region, on the basis of the existing primers EV1 and EV2, a pair of enterovirus universal primers EVN is designed again. Virus universal primer EVN comprises upstream primer EVN1 and downstream primer EVN2, wherein, the nucleotide sequence of upstream primer EVN1 is: 5'-ACTTCGAGAAGCCTAGTACC-3'; The sequence of downstream primer EVN2 is: 5'-TAGGATTAGCCGCATTCAG-3'; According to the comparison analysis on the GenBank database, the sequences with high similarity to the general primers of enterovirus are all derived from various enteroviruses, including poliovirus, coxsackie virus, echovirus, enterovirus type 71, No other microbial sequences with high similarity were found (Table 1).

Table 1: Sequences with high similarity to enterovirus universal primers

The following are the specific examples given by the inventor:

1. Equipment and reagents

(1) vacuum filter;

(2) High-speed refrigerated centrifuge with a maximum speed of 14000r/min;

(3) Centrifuge tube, 50mL, 1.5mL;

(4) UV spectrophotometer;

(5) Ultraviolet viewer;

(6) Constant temperature water bath box

(7) Real-time fluorescent quantitative PCR instrument;

(8) Poliovirus type 1;

(9) Vero cells;

(10) pMD18-T vector;

(11) Competent Escherichia coli XL1-blue:

(12) Mixed cellulose ester microporous filter membrane, with a pore size of 0.22 μm and a diameter of 50 mm;

(13) 2.5mol/L MgCl ₂ solution;

(14) 1mol/L hydrochloric acid;

(15) 3% beef extract-0.05mol/L glycine buffer solution (pH=9.5);

(16) polyethylene glycol, molecular weight 6000;

(17) 5mol/L NaCl solution;

(18) IPTG/X-gal/Amp plate;

(19) LB liquid medium;

(20) total RNA extraction kit;

(21) AMV reverse transcriptase;

(22) RNase Inhibitors

(23)dNTP mixture

(24)10×RT buffer

(25) SYBR Green I real-time fluorescent quantitative PCR kit;

(26) Enterovirus universal primer EV, including upstream primer EV1 and downstream primer EV2; the nucleotide sequence of upstream primer EV1 is: 5'-CAAGCACTTCTGTTTCCCCGG-3', and the nucleotide sequence of downstream primer EV2 is: 5'- ACCCATAGTAGTCGGTTCCGC-3'.

(27) Enterovirus universal primer EVN, including upstream primer EVN1 and downstream primer EVN2; the nucleotide sequence of upstream primer EVN1 is: 5'-ACTTCGAGAAGCCTAGTACC-3'; the nucleotide sequence of downstream primer EVN2 is: 5'- TAGGATTAGCCGCATTCAG-3', the amplified fragment is 231bp. Enterovirus universal primers are synthesized by professional bioengineering companies.

2. Preparation of Standards

The poliovirus was inoculated into Vero cells, and when the cell virus reached 3+-4+, the diseased cells were collected. Extract the RNA in the sample, use EV2 as a primer, add AMV reverse transcriptase, and perform reverse transcription according to a conventional method. Reverse transcription products were stored at -20°C.

Using the reverse transcription product as a template and EV1 and EV2 as primers, PCR amplification was carried out. The obtained target product fragment was recovered and purified, ligated with pMD18-T vector and transformed into Escherichia coli XL1-blue. Positive colonies were screened with IPTG/X-gal/Amp plates, cultured in LB liquid medium containing Amp, and plasmids were extracted for sequence determination. Quantify with an ultraviolet spectrophotometer, store at -20°C after gradient dilution, and use it as a standard for fluorescent real-time quantitative PCR.

Using recombinant plasmids with ten concentration gradients ranging from 2.31GEC/μL to 2.31×10 ⁹ GEC/μL as templates, real-time fluorescent quantitative PCR was carried out according to step (6) of the measurement procedure. Samples of each concentration were measured 5 times and the average value was taken. Regression, get the standard curve equation:

C _T ＝-3.6896logX ₀ +42.987(R ² ＝0.997) (Formula 1)

In the formula: X ₀ ——Initial template amount (GEC)

Enterovirus content in water samples: 40X ₀ (GEC) (Formula 2)

3. Measurement procedure

(1) Sample collection: Take 1L of water sample at 0.3m below the water surface, put it in an ice box at 4°C immediately after collection, and test within 6 hours.

(2) Preliminary concentration of virus: put the mixed cellulose ester filter membrane into the vacuum filter, and connect the vacuum pump. Take 1L of water sample (for water samples with more suspended particles, pre-filter with gauze first), add 2.5mol/L MgCl ₂ solution to make the final concentration of Mg ²⁺ 0.05mol/L, and then adjust the concentration of the solution with 1mol/L hydrochloric acid pH = 3.5, stir and mix on a magnetic stirrer. Filter through the filter membrane under negative pressure. After the filtration is completed, transfer the filter membrane to a clean beaker, add beef extract-glycine eluent (pH=9.5), fully infiltrate the filter membrane, then magnetically stir and elute for 30 minutes, discard the filter membrane, and collect the eluent.

(3) Secondary concentration of virus: add PEG6000 and 5mol/L NaCl to the eluent to make the final concentrations reach 130g/L and 0.2mol/L respectively, stir and mix well, and let stand overnight at 4°C. In a high-speed refrigerated centrifuge, centrifuge at 10000r/min, 4°C for 30min, discard the supernatant, and keep the precipitate.

(4) RNA extraction: use the total RNA extraction kit to extract the RNA in the precipitate according to the instructions, and finally make the volume to 52 μL.

(5) Reverse transcription: mix 13 μL RNA and 1 μL 25 μmol/L EV2 primer, add AMV reverse transcriptase, bathe in 70°C water for 10 minutes, ice bath for 10 minutes, and after centrifugation, add 2 μL 10×RTbuffer, 2 μL dNTP mixture (total 40 mmol /L), 10U RNase inhibitor and 5U AMV enzyme, in a water bath at 43°C for 1 hour, and in a water bath at 70°C for 10 minutes. Centrifuge briefly to obtain the reverse transcription product as a PCR template.

(6) Real-time fluorescent quantitative RT-PCR detection: Take 12.5 μL of 2×SYBR Mix, 0.5 μL each of 10 μmol/LEVN1 and 10 μmol/L EVN2, 2 μL of 25 mmol/L MgCl ₂ , 2 U of Taq enzyme, add 2 μL of template, and use a sterile ultra- Make up to 25 μL with pure water. Sterile ultrapure water was used as a negative control. Amplification and result analysis were carried out on a real-time quantitative PCR instrument, and the reaction conditions were:

① Pre-denaturation at 94°C for 4 minutes;

② Amplification cycle 40 times: 94°C, 30s; 55°C, 30s; 72°C, 30s;

③ Collect fluorescence signal at 85°C. Melting curve analysis was performed after the cycle was completed, and the temperature was raised from 65°C to 95°C at a rate of 0.2°C/s, and fluorescence was continuously detected during the whole process.

(7) Calculation: According to the _CT value obtained by the detection, it is substituted into the standard curve equation (formula 1) to calculate the initial template amount of enterovirus, and then substituted into formula 2 to calculate the content of enterovirus in the sample.

Compared with the qualitative RT-PCR detection technology of enteroviruses in existing environmental water bodies, the method of the present invention has the following significant advantages:

(1) Accurate quantification:

The present invention uses a series of gradient dilutions of recombinant plasmids as templates to carry out real-time fluorescent quantitative PCR to obtain the enterovirus standard curve (Fig. 1, 2), and the enterovirus content in the water sample can be quantified according to formula 1 and formula 2 detection.

(2) The sensitivity is greatly improved

The sensitivity of the existing qualitative RT-PCR detection technology for enteroviruses in environmental water is 38CCID ₅₀ , while the sensitivity of this method reaches 4.62GEC. (Although there is no clear quantitative relationship between CCID ₅₀ (cell culture median infectious dose and GEC (genome equivalent copy number)), CCID ₅₀ units are much greater than GEC)

(3) Wide linear range

The linear range of the usual real-time fluorescent quantitative RT-PCR detection method for viruses is 9 orders of magnitude, but when the template amount is in the range of 4.62 to 4.62×10 ⁹ GEC in this method, the logarithmic value of the template amount and _CT have a good linearity Relationship (R ² =0.997).

(4) High precision

The intra-assay coefficients of variation of the sample C _T values were all lower than 2%, and the inter-assay coefficients of variation were all lower than 5%, indicating that the method has high precision (Table 2 and Table 3).

Table 2: Intra-assay coefficient of variation of test samples

Table 3: Coefficient of variation between batches of test samples

(5) has good specificity

There is no cross-reaction with other pathogenic microorganisms commonly found in the environment.

Claims (1)

1. A quantitative detection method for enteroviruses in environmental water, characterized in that, comprises the following steps: Step 1, enterovirus universal primer EVN design: According to the homology of the conserved sequence of the enterovirus RNA 5' non-coding region, on the basis of the existing primers EV1 and EV2, a pair of enterovirus universal primers EVN is designed, and the enterovirus universal primer EVN includes the upstream primer EVN1 and the downstream primer EVN2, wherein the nucleotide sequence of the upstream primer EVN1 is: 5'-ACTTCGAGAAGCCTAGTACC-3'; the sequence of the downstream primer EVN2 is: 5'-TAGGATTAGCCGCATTCAG-3'; The nucleotide sequence of the primer EV1 is: 5'-CAAGCACTTCTGTTTCCCCGG-3', and the nucleotide sequence of the primer EV2 is: 5'-ACCCATAGTAGTCGGTTCCGC-3'; Step 2, preparation of standard substance: Inoculate the poliovirus into Vero cells, when the cell virus reaches 3+~4+, collect the diseased cells, extract the RNA in the sample, use EV2 as a primer, add AMV reverse transcriptase for reverse transcription, and the reverse transcription product Store at -20°C; Using reverse transcription product as template, EV1 and EV2 as primers, carry out PCR amplification, recover and purify the obtained target product fragment, connect with pMD18-T vector and transform into E. Cultivate in LB liquid medium, extract the plasmid for sequence determination, quantify it with an ultraviolet spectrophotometer, store it at -20°C after gradient dilution, and use it as a standard for fluorescent real-time quantitative PCR; Use recombinant plasmids with ten concentration gradients ranging from 2.31GEC/μL to 2.31×10 ⁹ GEC/μL as templates, where GEC is the genome equivalent copy number, perform real-time fluorescent quantitative PCR according to the measurement procedure, and measure 5 times for each concentration of samples. Take the mean value for regression to get the standard curve equation: C _T ＝-3.6896logX ₀ +42.987R ² ＝0.997 Formula 1 In the formula: X ₀ ——Initial template amount GEC Enterovirus content in water samples: 40X ₀ GEC Formula 2 Step three, quantitative detection: 1) Sample collection: Take water samples at 0.3m below the water surface, and store them in a 4°C ice box after collection; 2) Preliminary concentration of virus: the mixed cellulose ester filter membrane is packed into a vacuum filter, the water sample containing enterovirus is filtered, the membrane is eluted with beef extract-glycine buffer as the eluent, and the eluate is collected; 3) Secondary concentration: add PEG and NaCl to the eluent, let it stand overnight at 4°C, stir and mix well, let stand at 4°C to precipitate overnight, centrifuge, and collect the precipitate; 4) RNA extraction: use the RNA extraction kit to extract viral RNA from the precipitate; 5) Reverse transcription: Mix 13 μL RNA and 1 μL 25 μmol/L EV2 primer, add AMV reverse transcriptase, bathe in 70°C water for 10 minutes, ice bath for 10 minutes, centrifuge briefly, add 2 μL 10×RTbuffer, 2 μL dNTP mixture to a total of 40 mmol/L , 10U RNase inhibitor and 5U AMV enzyme, 43 ° C water bath for 1 h, 70 ° C water bath for 10 min, centrifuged, and the reverse transcription product was obtained as a PCR template; 6) Real-time fluorescent quantitative RT-PCR detection: Take 12.5 μL of 2×SYBR Mix, 0.5 μL each of 10 μmol/L upstream primer EVN1 and 10 μmol/L downstream primer EVN2, 2 μL 25 mmol/L MgCl ₂ , Taq enzyme 2U, add 2 μL of template, Make up to 25 μL with sterile ultrapure water, use sterile ultrapure water as a template as a negative control, and perform amplification and result analysis on a real-time quantitative PCR instrument; The reaction conditions are: ① Pre-denaturation at 94°C for 4 minutes; ② Amplification cycle 40 times: 94°C, 30s; 55°C, 30s; 72°C, 30s; ③Collect fluorescence signal at 85°C, perform melting curve analysis after the cycle ends, heat up from 65°C to 95°C at a rate of 0.2°C/s, and continuously detect fluorescence during the whole process; 7) Calculation: According to the _CT value obtained from the detection, it is substituted into formula 1 of the standard curve equation to calculate the initial template amount of enterovirus, and then substituted into formula 2 to calculate the content of enterovirus in the sample.

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