CN102888469A - Method for detecting content of human enteroviruses and non-human enteroviruses in environmental water - Google Patents

Abstract

The invention discloses a method for detecting content of human enteroviruses and non-human enteroviruses in environmental water. On the basis of the established method for quantitatively detecting the enteroviruses in the environmental water, primers and probes for the human enteroviruses are selected, recombinant plasmids are prepared as standard substances, a real-time fluorescent quantitative PCR reaction system for quantitatively detecting the human enteroviruses is established, the content of the human enteroviruses and the enteroviruses in environmental water is quantitatively detected to further determine a distribution condition of the non-human enteroviruses. The method has good specificity and high sensitivity, accurately quantifies the human enteroviruses and the enteroviruses and determines relationship between the human enteroviruses and the non-human enteroviruses in the environmental water.

Description

Human intestine's virus and non-human enterovirus detection method of content in the environment water

Technical field

The invention belongs to the detection technique field of environment water quality, be specifically related to human intestine's virus and non-human enterovirus detection method of content in a kind of environment water.

Background technology

Enterovirus is extensively to be present in source water and the polluted water, constantly causes the outbreak of epidemic of hydrophily viral disease.The Detecting method mainly contains: tissue culture method, immunological method, molecular biology method etc.Wherein, the tissue culture method culture cycle is long, and some virus lacks the clone of sensitivity and cultivates or the obvious cytopathic effect of nothing; Immunological detection method needs the viral sample of higher concentration, after environmental water sample is concentrated, also is difficult for reaching desired concn; Molecular biology method utilizes the detection of nucleic acid more, because of its detection time short, the detectability muting sensitivity is high, tolerance range is high, is widely used.

It is totally different that Enteroviruses from Environmental Waters belongs to the host, the complicated concentration of kind is low, existing PCR detection method about Enteroviruses from Environmental Waters is the qualitative or quantitative total enterovirus of detection mostly, and the detection that derives from human secretory product or excremental enterovirus seldom has research to relate to.Because environment water water quality complexity exists a lot of PCR reaction suppressors and other impurity simultaneously, cause existing detection method exist insufficient sensitivity high, be subjected to that the impurity disturbing influence is large, shortcoming that can't accurate quantitative analysis.The method viral to the environment water human intestine and that non-human enterovirus content detects that is used for that does not also have at present science.

Summary of the invention

Technical problem to be solved by this invention is to provide human intestine's virus and non-human enterovirus detection method of content in a kind of environment water.

For this reason, human intestine's virus is carried out in the steps below with non-human enterovirus detection method of content in the environment water provided by the invention:

Step 1, sample preparation:

(1) sample collecting: under 4 ℃ of conditions, preserve after gathering water sample to be detected;

(2) virus is concentrated: add mass percent and be 8% polyoxyethylene glycol (PEG-6000) and mass percent in the water sample to be detected of V mL and be 2.3% sodium-chlor (NaCl); Then water sample to be detected was stirred 12 hours under 100rpm, 4 ℃ of conditions; Then with water sample to be detected at 9000g, under 4 ℃ of conditions centrifugal 30 minutes, abandon supernatant liquor, obtain viral concentrated solution with the resuspended precipitation of 1mL ultrapure water, wherein 50≤V≤250;

(3) RNA extracts: use RNA to extract test kit and extract RNA from viral concentrated solution;

(4) reverse transcription: with the 5xgDNA Eraser Buffer of 5xgDNA Eraser Buffer, the 1 μ L of RNA, the 2 μ L of 5 μ L and the RNase Free dH of 2 μ L ₂O reacts 2min and obtains reaction solution under 42 ℃ of conditions, with this reaction solution and 4 μ L

Buffer, 1 μ L's The RNase Free dH of the RT Primer Mix of RT Enzyme Mix, 1 μ L and 4 μ L ₂Following reaction is carried out in the O mixing: react 15min first under 37 ℃ of conditions; Then under 85 ℃ of conditions, react 5s; Obtain cDNA, and gained cDNA is preserved under-80 ℃ of conditions; Step 2, the standard substance preparation:

(1) first round pcr amplification: the cDNA that obtains take step 1 utilizes upstream primer EVR-F1 and downstream primer EVR-R1 to carry out pcr amplification as template, obtains the PCR product after the first round amplification, wherein:

The nucleotides sequence of upstream primer EVR-F1 is classified as:

5’-CCCCTGAATGCGGCTAATCC-3’；

The nucleotides sequence of downstream primer EVR-R1 is classified as:

5’-CAATTGTCACCATAAGCAGCCA-3’；

(2) second take turns pcr amplification: the PCR product after the first round amplification utilizes upstream primer EVR-F2 and downstream primer EVR-R2 to carry out pcr amplification as template, obtains the second PCR product of taking turns after the amplification, wherein:

The nucleotides sequence of upstream primer EVR-F2 is classified as:

5’-GTAACGGGCAACTCTGCAGC-3’；

The nucleotides sequence of downstream primer EVR-R2 is classified as:

5’-ATTGTCACCATAAGCAGCCA-3’；

The PCR product running gel purifying of (3) second of gained being taken turns after the amplification reclaims the purpose fragment, obtains purpose fragment product, and the length of this purpose fragment product is m(bp);

(4) at first purpose fragment product is connected with the pMD19-T carrier and is converted in the bacillus coli DH 5 alpha, filter out the bacterium colony of the carrier that transforms to advance to insert the purpose fragment, this positive bacteria cultivated under 37 ℃ of conditions in containing the LB liquid nutrient medium of kantlex obtained bacterium liquid in 12 ~ 16 hours, wherein the mass concentration of kantlex is 50 μ g/mL; Then from bacterium liquid, extract plasmid with plasmid extraction kit; Then adopt two deoxidation cessation method that the nucleotide sequence of the plasmid that obtains is measured, measured nucleotides sequence is listed in the NCBI gene pool to be compared through BLAST, when measured nucleotide sequence and human intestine's virogene similarity more than or equal to 90% the time, the plasmid of extraction is standard substance; When measured nucleotide sequence and human intestine's virogene similarity less than 90% the time, repeating step (4) is until the plasmid that extracts is standard substance;

(5) OD of examination criteria product ₂₆₀Value n;

(6) utilize (formula 1) to calculate standard substance concentration C (copies/ μ L):

$C=\frac{{n\×50\×10}^{-9}}{(2962+m)\×2\×330}\×6.02\×{10}^{23}$ (formula 1)

In the formula 1:

2962 is the length of pMD19-T carrier, bp;

1OD ₂₆₀The double-stranded DNA of=50 μ g/mL;

330 is the molecular weight of 1 base, g/mol;

6.02 * 10 ²³Be Avogadro constant, NA;

(7) preparation cx10 ^-4Copies/ μ L, cx10 ^-5Copies/ μ L, cx10 ^-6Copies/ μ L, cx10 ^-7Copies/ μ L, cx10 ^-8Copies/ μ L, cx10 ^-9Copies/ μ L and cx10 ^-10The standard solution sample of seven concentration gradients of copies/ μ L, utilize real-time fluorescence quantitative PCR to detect respectively Ct value and the initial masterplate amount x(copies of each concentration gradient sample), then utilize seven groups of Ct-x that record to carry out linear regression analysis, obtain the typical curve equation:

Ct=alogx+b (formula 2)

In the formula 2: a and b are the coefficient of typical curve equation;

Step 3, detection by quantitative:

(1) with the 2xPremix EX Taq of 10 μ L ^TMThe cDNA that the TaqMan probe EVR-P of downstream primer EVR-R2, the 400nM of upstream primer EVR-F2, the 200nM of Mix, 200nM, the 50xROX Reference Dye II of 0.4 μ L and 2 μ L step 1 obtain mixes, then complement to 20 μ L with aseptic ultrapure water, increase the Ct value Ct of the cDNA that detecting step one obtains at real-time PCR ₀, simultaneously with aseptic ultrapure water as negative control; PCR reaction conditions wherein is: 1. 95 ℃ of denaturation 2min; 2. amplification cycles is 45 times: 95 ℃, and 15s; 60 ℃, 60s;

The nucleotides sequence of described TaqMan probe EVR-P is classified as:

5'-6-FAM-CACGGACACCCAAAGTAGTCGGTTCC-BHQ1；

(2) with Ct ₀The original template amount x of water sample to be detected is calculated in substitution (formula 2) ₀, the content C of human intestine's virus in the water sample to be detected _HEV(copies/mL) be:

$C_{\mathrm{HEV}}=\frac{x_0(20/2)(60/5)(1000/140)}{V}$ (formula 3)

(3) detect the content C of enterovirus in the water sample to be detected according to the quantitative detecting method of Enteroviruses from Environmental Waters _EV(copies/mL);

(4) non-human enterovirus content C in the water sample to be detected _M(copies/mL) be:

C _M=C _EV-C _HEV(formula 4).

The OD of step 2 (6) Plays product ₂₆₀Value adopts micro-UV spectrophotometer measuring.

But the content of human intestine's virus in the detection method accurate quantitative analysis testing environment water body of the present invention does not have cross reaction with other pathogenic micro-organisms common in the environment, has higher sensitivity and precision.

Be used for primer EVR-F2, the EVR-R2 of human intestine's virus detection by quantitative and the nucleotide sequence of probe EVR-P, have high similarity with the gene that derives from human intestine's virus, do not find the enterovirus in other animality sources that similarity is higher.Simultaneously, utilize probe EVR-P as the fluorescent signal report group, EVR-P can be specific and the pairing of human intestine's virogene base complementrity, avoided the interference of non-target gene, increased specificity and the accuracy of detection method.

Description of drawings

Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail.

Fig. 1 is the typical curve of embodiment.

Embodiment

Reverse transcription polymerase chain reaction (RT-PCR) technology is carried out reverse transcription take RNA as template, and can with the RNA of denier a few hours internal specific be expanded to up to a million times.

Real-time fluorescence quantitative RT-PCR utilizes fluorescent signal to come the whole PCR process of Real-Time Monitoring by add fluorophor in reaction system, and the typical curve that uses at last the concentration known sample to draw carries out quantitative analysis to unknown template concentrations.

Existing quantitative PCR detection is mainly used in medical field, and test samples is mainly from people's secretory product or movement, and the enterovirus that detects is human enterovirus substantially.

The contriver is on the basis of the detection method of existing Enteroviruses from Environmental Waters, homology according to enteroviral rna 5 ' non-coding region conserved sequence, select one group specifically for the special primer EVR-F2 of human intestine's virus, EVR-R2 and TaqMan probe EVR-P, wherein upstream primer EVR-F2 nucleotides sequence is classified as: 5'-GTAACGGGCAACTCTGCAGC-3', downstream primer EVR-R2 nucleotides sequence is classified as: 5'-ATTGTCACCATAAGCAGCCA-3', TaqMan probe EVR-P nucleotides sequence is classified as: 5'-6-FAM-CACGGACACCCAAAGTAGTCGGTTCC-BHQ1.

The nucleic acid fragment that comprises in order from environment water, to obtain EVR-F2 and EVR-R2, select again in addition pair of primers EVR-F1:5'-CCCCTGAATGCGGCTAATCC-3' and EVR-R1:5'-CAATTGTCACCATAAGCAGCCA-3' to consist of nest-type PRC and increase, in order in the very low environment water of concentration, obtain the purpose fragment;

At NCBI gene pool compare of analysis, the sequence that has high similarity with the selected human intestine's virus primer of the present invention and probe all derives from human intestine's virus, does not find other microorganism sequence that similarity is higher through BLAST.

Embodiment:

Equipment and reagent that this embodiment is used are as follows:

(1) high speed freezing centrifuge, maximum speed of revolution 14000r/min;

(2) 4 ℃ ,-80 ℃ refrigerators

(3) centrifuge tube, 50mL, 1.5mL, 0.5mL, 0.2mL;

(4) micro-ultraviolet spectrophotometer;

(5) thermostat metal well heater;

(6) bacterium shaking table, bacteriological incubator;

(7) real-time fluorescence quantitative PCR instrument;

(8) gradient qualitative PCR instrument;

(9) electrophoresis apparatus;

(10) gel imaging instrument;

(11) pMD19-T carrier;

(12) competence bacillus coli DH 5 alpha;

(13) hydrochloric acid, NaCl solution, MgCl ₂Solution etc.;

(14) polyoxyethylene glycol of molecular weight 6000 (PEG-6000), glucose etc.;

(15) 5mol/L NaCl solution;

(16) IPTG/X-gal/Amp is dull and stereotyped;

(17) LB liquid nutrient medium;

(18) total RNA extraction reagent box;

(19) reverse transcription test kit;

（20）dNTP?mixture；

（21）10×Ex?Taq?buffer；

(22) Premix Ex Taq ^TMThe real-time fluorescence quantitative PCR test kit;

(23) plasmid extraction kit;

(24) human intestine's virus-specific primer EVR-F1:5'-CCCCTGAATGCGGCTAATCC-3',

EVR-R1：5'-CAATTGTCACCATAAGCAGCCA-3'，EVR-F2：

5'-GTAACGGGCAACTCTGCAGC-3'，EVR-R2：

5'-ATTGTCACCATAAGCAGCCA-3', and TaqMan probe EVR-P:

5'-6-FAM-CACGGACACCCAAAGTAGTCGGTTCC-BHQ1 is synthetic by Sangon Biotech (Shanghai) Co., Ltd..

(25) enterovirus universal primer EV1:5'-CAAGCACTTCTGTTTCCCCGG-3', EV2:

5′-ACCCATAGTAGTCGG?T?T?CCGC-3'，EVN1：

5'-ACTTCGAGAAGCCTAGTACC-3′，EVN2：

5'-TAGGATTAGCCGCATTCAG-3 ', synthetic by Sangon Biotech (Shanghai) Co., Ltd..

Follow technical scheme of the present invention, the concrete grammar step of this embodiment is as follows:

Step 1, sample preparation:

(1) sample collecting: gather Xi'an Sewage Plant raw waste water, under 4 ℃ of conditions, preserve;

(2) virus is concentrated: the PEG-6000 that in the water sample to be detected of 50mL, adds 4g make its mass concentration be 8% and the NaCl of 1.15g to make its mass concentration be 2.3%, follow water sample to be detected at 100rpm, stirred 12 hours under 4 ℃ of conditions, then with water sample to be detected at 9000g, under 4 ℃ of conditions centrifugal 30 minutes, abandon supernatant liquor, obtain viral concentrated solution with the resuspended precipitation of 1mL ultrapure water;

(3) RNA extracts: use RNA to extract test kit and extract RNA from viral concentrated solution;

(4) reverse transcription: with the 5xgDNA Eraser Buffer of 5xgDNA Eraser Buffer, the 1 μ L of RNA, the 2 μ L of 5 μ L and the RNase Free dH of 2 μ L ₂O reacts 2min and obtains reaction solution under 42 ℃ of conditions, with this reaction solution and 4 μ L

Buffer, 1 μ L's The RNase Free dH of the RT Primer Mix of RT Enzyme Mix, 1 μ L and 4 μ L ₂Following reaction is carried out in the O mixing: react 15min first under 37 ℃ of conditions; Then under 85 ℃ of conditions, react 5s; Obtain cDNA, and gained cDNA is preserved under-80 ℃ of conditions;

Step 2, the standard substance preparation:

(1) first round pcr amplification: take cDNA that step 1 was obtained as template, utilize upstream primer EVR-F1 and downstream primer EVR-R1 to carry out pcr amplification, obtain the PCR product after the first round amplification,

(2) second take turns pcr amplification: the PCR product after the first round amplification utilizes upstream primer EVR-F2 and downstream primer EVR-R2 to carry out pcr amplification as template, obtains the second PCR product of taking turns after the amplification,

The PCR product running gel purifying of (3) second of gained being taken turns after the amplification reclaims the purpose fragment, obtains purpose fragment product, the length m=85bp of this purpose fragment product;

(4) purpose fragment product is connected with the pMD19-T carrier is converted in the intestinal bacteria, filter out the positive bacterium colony of cloning successfully, this positive bacteria cultivated under 37 ℃ of conditions in containing the LB liquid nutrient medium of Amp obtained bacterium liquid in 16 hours, then from 2mL bacterium liquid, extract plasmid with plasmid extraction kit;

(5) adopt two deoxidation cessation method that the nucleotide sequence of the plasmid that obtains is measured, measured nucleotide sequence is compared through BLAST at the NCBI gene pool, when measured nucleotide sequence and human intestine's virogene similarity more than or equal to 90% the time, the plasmid of extraction is standard substance; When measured nucleotide sequence and human intestine's virogene similarity less than 90% the time, repeating step (4) and step (5) are until the plasmid that extracts is standard substance; With standard substance in-80 ℃ of Refrigerator stores;

(6) with the OD of micro-UV spectrophotometer measuring standard substance ₂₆₀Value n=1.342;

(7) utilizing (formula 1) to calculate the standard substance concentration C is 2.016 * 10 ¹⁰Copies/ μ L:

(8) standard substance are diluted to 2.016 * 10 successively ⁶Copies/ μ L, 2.016 * 10 ⁵Copies/ μ L, 2.016 * 10 ⁴Copies/ μ L, 2.016 * 10 ³Copies/ μ L, 2.016 * 10 ²Copies/ μ L, 2.016 * 10 ¹Copies/ μ L and 2.016 * 10 ⁰Seven concentration gradients of copies/ μ L utilize real time fluorescence quantifying PCR method to detect respectively the Ct value of each concentration gradient sample, i.e. different original template amount x(copies) the Ct value, then utilize seven groups of Ct-x{(4.032 * 10 that record ⁶, 18.042), (4.032 * 10 ⁵, 21.597), (4.032 * 10 ⁴, 24.951), (4.032 * 10 ³, 28.434), (4.032 * 10 ², 32.053), (4.032 * 10 ¹, 34.741), (4.032 * 10 ⁰, 37.984) }, carry out linear regression analysis, obtain typical curve shown in Figure 1, this typical curve equation is:

Ct=-3.329logx+40.261 (formula 5)

Step 3, detection by quantitative:

(1) with the 2xPremix EX Taq of 10 μ L ^TMThe concentration of Mix, 0.4 μ L is that the concentration of upstream primer EVR-F2, the 0.4 μ L of 10 μ mol/L is that the concentration of 10 μ mol/L downstream primer EVR-R2,0.5 μ L is that the 50xROX Reference Dye II of 10 μ mol/L TaqMan probe EVR-P, 0.4 μ L and cDNA that 2 μ L step 1 obtain mix, then complement to 20 μ L with aseptic ultrapure water, increase at real-time PCR, simultaneously make negative control with aseptic ultrapure water, the Ct value Ct of the cDNA that detecting step one obtains ₀Be 32.271; PCR reaction conditions wherein is: 1. 95 ℃ of denaturation 2min; 2. amplification cycles is 45 times: 95 ℃, and 15s; 60 ℃, 60s;

(2) with Ct ₀The original template amount x of raw waste water is calculated in substitution (formula 5) ₀Be 2.512 * 10 ²Copies, the content C of human intestine's virus in the water sample to be detected _HEVCalculate with (formula 3), human intestine's viral level is 4.306 * 10 in the raw waste water ³Copies/mL:

(3) utilize the quantitative detecting method (quantitative detecting method of the disclosed Enteroviruses from Environmental Waters of ZL200810150350.0) of Enteroviruses from Environmental Waters to detect the content C of enterovirus in the water sample to be detected _EVBe 5.277 * 10 ³Copies/mL;

(4) non-human enterovirus content C in the water sample to be detected _MBe 9.710 * 10 ²Copies/mL.

The contriver verifies the precision of the quantitative PCR detecting method among the present invention, chooses the cDNA of a concentration known sample, carries out 6 real-time fluorescence quantitative PCRs and detect in same batch, calculates the group within variance coefficient of Ct value; Then divide 6 batches selected sample is carried out 6 times to detect, calculate the between-group variation coefficient of Ct.Group within variance coefficient and the between-group variation coefficient of table 1 for detecting, the group within variance coefficient of sample Ct value all is lower than 2%, and the between-group variation coefficient all is lower than 3%, illustrates that present method has higher precision.

In the group of table 1 quantitative PCR detecting method and the between-group variation coefficient

Claims (2)

1. human intestine's virus and non-human enterovirus detection method of content in the environment water is characterized in that, method is carried out in the steps below:

Step 1, sample preparation:

(1) sample collecting: under 4 ℃ of conditions, preserve after gathering water sample to be detected;

(2) virus is concentrated: add mass percent and be 8% polyoxyethylene glycol (PEG-6000) and mass percent in the water sample to be detected of V mL and be 2.3% sodium-chlor (NaCl); Then water sample to be detected was stirred 12 hours under 100rpm, 4 ℃ of conditions; Then with water sample to be detected at 9000g, under 4 ℃ of conditions centrifugal 30 minutes, abandon supernatant liquor, obtain viral concentrated solution with the resuspended precipitation of 1mL ultrapure water, wherein 50≤V≤250;

(3) RNA extracts: use RNA to extract test kit and extract RNA from viral concentrated solution;

(4) reverse transcription: with 5 * gDNA Eraser Buffer of 5 * gDNA Eraser Buffer, the 1 μ L of RNA, the 2 μ L of 5 μ L and the RNase Free dH of 2 μ L ₂O reacts 2min and obtains reaction solution under 42 ℃ of conditions, with this reaction solution and 4 μ L

Buffer, 1 μ L's The RNase Free dH of the RT Primer Mix of RT Enzyme Mix, 1 μ L and 4 μ L ₂Following reaction is carried out in the O mixing: react 15min first under 37 ℃ of conditions; Then under 85 ℃ of conditions, react 5s; Obtain cDNA, and gained cDNA is preserved under-80 ℃ of conditions;

Step 2, the standard substance preparation:

(1) first round pcr amplification: the cDNA that obtains take step 1 utilizes upstream primer EVR-F1 and downstream primer EVR-R1 to carry out pcr amplification as template, obtains the PCR product after the first round amplification, wherein:

The nucleotides sequence of upstream primer EVR-F1 is classified as:

5’-CCCCTGAATGCGGCTAATCC-3’；

The nucleotides sequence of downstream primer EVR-R1 is classified as:

5’-CAATTGTCACCATAAGCAGCCA-3’；

(2) second take turns pcr amplification: the PCR product after the first round amplification utilizes upstream primer EVR-F2 and downstream primer EVR-R2 to carry out pcr amplification as template, obtains the second PCR product of taking turns after the amplification, wherein:

The nucleotides sequence of upstream primer EVR-F2 is classified as:

5’-GTAACGGGCAACTCTGCAGC-3’；

The nucleotides sequence of downstream primer EVR-R2 is classified as:

5’-ATTGTCACCATAAGCAGCCA-3’；

The PCR product running gel purifying of (3) second of gained being taken turns after the amplification reclaims the purpose fragment, obtains purpose fragment product, and the length of this purpose fragment product is m(bp);

(4) at first purpose fragment product is connected with the pMD19-T carrier and is converted in the bacillus coli DH 5 alpha, filter out the bacterium colony of the carrier that transforms to advance to insert the purpose fragment, this positive bacteria cultivated under 37 ℃ of conditions in containing the LB liquid nutrient medium of kantlex obtained bacterium liquid in 12 ~ 16 hours, wherein the mass concentration of kantlex is 50 μ g/mL; Then from bacterium liquid, extract plasmid with plasmid extraction kit; Then adopt two deoxidation cessation method that the nucleotide sequence of the plasmid that obtains is measured, measured nucleotides sequence is listed in the NCBI gene pool to be compared through BLAST, when measured nucleotide sequence and human intestine's virogene similarity more than or equal to 90% the time, the plasmid of extraction is standard substance; When measured nucleotide sequence and human intestine's virogene similarity less than 90% the time, repeating step (4) is until the plasmid that extracts is standard substance;

(5) OD of examination criteria product ₂₆₀Value n;

(6) utilize (formula 1) to calculate standard substance concentration C (copies/ μ L):

$C=\frac{n\×50\×{10}^{-9}}{(2962+m)\×2\×330}\×6.02\×{10}^{23}$ (formula 1)

In the formula 1:

2962 is the length of pMD19-T carrier, bp;

1OD ₂₆₀The double-stranded DNA of=50 μ g/mL;

330 is the molecular weight of 1 base, g/mol;

6.02 * 10 ²³Be Avogadro constant, NA;

(7) preparation c * 10 ^-4Copies/ μ L, c * 10 ^-5Copies/ μ L, c * 10 ^-6Copies/ μ L, c * 10 ^-7Copies/ μ L, c * 10 ^-8Copies/ μ L, c * 10 ^-9Copies/ μ L and c * 10 ^-10The standard solution sample of seven concentration gradients of copies/ μ L, utilize real-time fluorescence quantitative PCR to detect respectively Ct value and the initial masterplate amount x(copies of each concentration gradient sample), then utilize seven groups of Ct-x that record to carry out linear regression analysis, obtain the typical curve equation:

Ct=alogx+b (formula 2)

In the formula 2: a and b are the coefficient of typical curve equation;

Step 3, detection by quantitative:

(1) with 2 * Premix EX Taq of 10 μ L ^TMThe cDNA that the TaqMan probe EVR-P of downstream primer EVR-R2, the 400nM of upstream primer EVR-F2, the 200nM of Mix, 200nM, 50 * ROXReference Dye II of 0.4 μ L and 2 μ L step 1 obtain mixes, then complement to 20 μ L with aseptic ultrapure water, increase the Ct value Ct of the cDNA that detecting step one obtains at real-time PCR ₀, simultaneously with aseptic ultrapure water as negative control; PCR reaction conditions wherein is: 1. 95 ℃ of denaturation 2min; 2. amplification cycles is 45 times: 95 ℃, and 15s; 60 ℃, 60s;

The nucleotides sequence of described TaqMan probe EVR-P is classified as:

5'-6-FAM-CACGGACACCCAAAGTAGTCGGTTCC-BHQ1；

(2) with Ct ₀The original template amount x of water sample to be detected is calculated in substitution (formula 2) ₀, the content C of human intestine's virus in the water sample to be detected _HEV(copies/mL) be:

$C_{\mathrm{HEV}}=\frac{x_0(20/2)(60/5)(1000/140)}{V}$ (formula 3)

(3) detect the content C of enterovirus in the water sample to be detected according to the quantitative detecting method of Enteroviruses from Environmental Waters _EV(copies/mL);

(4) non-human enterovirus content C in the water sample to be detected _M(copies/mL) be:

C _M=C _EV-C _HEV(formula 4).

2. human intestine's virus and non-human enterovirus detection method of content in the environment water as claimed in claim 1 is characterized in that the OD of step 2 (5) Plays product ₂₆₀Value adopts micro-UV spectrophotometer measuring.

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