CN101333568B - Quantitative determination process for enterovirus in environment water body - Google Patents
Quantitative determination process for enterovirus in environment water body Download PDFInfo
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Abstract
The invention discloses a quantitative detection method of enteroviruses in environmental water, according to the homology of a conserved sequence at an RNA5` non-coding region of enteroviruses and on the basis of the existing primers of EV1 and EV2, the method further designs a pair of enterovirus universal primers EVN, prepares a series of recombinant plasmids adopting gradient dilution as standard substances, and builds a real-time fluorescence quantitative PCR reaction system applying the enterovirus universal primers EVN, so as to quantitatively detect the enterovirus content in the environmental water. The method has advantages of accurate quantification, greatly improving the sensitivity of the enterovirus qualitative RT-PCR detection technique in the current environmental water, broad linearity range, high precision, good specificity, and having no cross reaction with other common pathogenic microorganisms in the environment.
Description
Technical field
The present invention relates to the quantitative detecting method of enterovirus in a kind of environment water, this method is measured enterovirus with real-time fluorescent quantitative RT-PCR method, is suitable for the quantitative assay of the multiple virus of enterovirus genus in the environment water.
Background technology
Reverse transcription polymerase chain reaction (RT-PCR) technology is article one chain that becomes complementary DNA with RNA for the template reverse transcription, is that template is carried out PCR reaction with this chain again, thus can be with the RNA of denier up to a million times of a few hours internal specific amplifications.The real-time fluorescence quantitative RT-PCR technology utilizes fluorescent signal to monitor whole PCR process in real time by add fluorophor in reaction system, uses typical curve that unknown template concentrations is carried out quantitative analysis at last.The existing method that detects about enteron aisle virus PCR in the environment water is based on qualitative RT-PCR detection technique mostly, and existence can't be accurately quantitatively, insufficient sensitivity is high, be subjected to the bigger shortcoming of impurity disturbing influence.Though the real-time fluorescence quantitative RT-PCR technology has been applied to the microorganism detection of medical field, can not be fit to the needs of environment water, therefore also there is not at present a cover to be applicable to the real-time fluorescent quantitative RT-PCR method that the environment water enterovirus detects.
Summary of the invention
Defective or deficiency at above-mentioned prior art exists the objective of the invention is to, and the quantitative detecting method of enterovirus in a kind of environment water is provided, and with the problem of solution detection by quantitative, and improve sensitivity and the specificity that detects.
In order to realize above-mentioned task, the present invention takes following technical solution:
The quantitative detecting method of enterovirus is characterized in that in a kind of environment water, comprises the following steps:
Step 1, enterovirus universal primer EVN design:
Homology according to enteroviral rna 5 ' non-coding region conserved sequence, on the basis of existing primer EV1 and EV2, design a pair of enterovirus universal primer EVN again, described enterovirus universal primer EVN comprises upstream primer EVN1 and downstream primer EVN2, wherein, the nucleotides sequence of upstream primer EVN1 is classified as: 5 '-ACTTCGAGAAGCCTAGTACC-3 '; The sequence of downstream primer EVN2 is: 5 '-TAGGATTAGCCGCATTCAG-3 ';
Poliovirus is inoculated in the Vero cell, when cell virus reaches 3+～4+, collects sick cell, extracting the RNA in the sample, is primer with EV2, adds the AMV reversed transcriptive enzyme, carry out reverse transcription according to ordinary method, the reverse transcription product places-20 ℃ of preservations;
With the reverse transcription product is template, and EV1 and EV2 are primer, carry out pcr amplification, and the purpose product fragment of gained is reclaimed purifying, is connected with the pMD18-T carrier to be converted in the intestinal bacteria.Select positive bacteria with flat screen and fall, cultivate in containing the LB liquid nutrient medium of Amp, extract plasmid and carry out sequencing, quantitative with ultraviolet spectrophotometer again ,-20 ℃ of preservations are as the standard substance of fluorescence real-time quantitative PCR behind the gradient dilution;
Utilize 2.31GEC/ μ L～2.31 * 10
9The recombinant plasmid of ten concentration gradients of GEC/ μ L carries out real-time fluorescence quantitative PCR as template according to the mensuration program, the sample determination of each concentration 5 times, and averaging returns, and obtains the typical curve equation:
C
T=-3.6896logX
0+ 42.987 (R
2=0.997) (formula 1)
In the formula: X
0---original template amount (GEC)
Enterovirus content in the water sample: 40X
0(GEC) (formula 2)
Step 3, detection by quantitative:
1) sample collecting:, put into 4 ℃ of ice chests after the water sampling and preserve at underwater 0.3m place water sampling;
2) the preliminary of virus concentrates: with the mixed cellulose ester filter membrane vacuumfilter of packing into, filter the water sample that contains enterovirus, carry out membrane elution with extractum carnis-glycine buffer as eluent, the collection elutriant;
3) secondary concentration: add PEG and NaCl in elutriant, 4 ℃ of standing over night stirring and evenly mixings staticly settle under 4 ℃ and spend the night, and are centrifugal, collecting precipitation;
4) RNA extracts: utilize RNA to extract test kit, extract viral RNA from precipitation;
5) reverse transcription: with 13 μ L RNA and 1 μ L, 25 μ mol/L EV2 primer mixings, add the AMV reversed transcriptive enzyme, 70 ℃ of water-bath 10min, ice bath 10min, instantaneous centrifugal after, add 2 μ L, 10 * RTbuffer, 2 μ L dNTP mixture (40mmol/L altogether), 10U RNA enzyme inhibitors and 5U AMV enzyme, 43 ℃ of water-bath 1h, 70 ℃ of water-bath 10min, centrifugal, obtain the reverse transcription product as pcr template;
6) real-time fluorescence quantitative RT-PCR detects: get 2 * SYBR Mix, 12.5 μ L, each 0.5 μ L of 10 μ mol/L upstream primer EVN1 and 10 μ mol/L downstream primer EVN2,2 μ L 25mmol/L MgCl
2, Taq enzyme 2U adds template 2 μ L, complements to 25 μ L with aseptic ultrapure water, does the negative contrast of template with aseptic ultrapure water, increases and interpretation of result on the real-time quantitative PCR instrument;
Reaction conditions is:
1. 94 ℃ of pre-sex change 4min;
2. amplification cycles is 40 times: 94 ℃, and 30s; 55 ℃, 30s; 72 ℃, 30s;
3. collect fluorescent signal at 85 ℃, carry out the melting curve analysis after the loop ends, be warming up to 95 ℃, continue to detect fluorescence in the whole process from 65 ℃ with 0.2 ℃/s speed;
7) calculate: according to the C that detects gained
TBe worth, calculate the original template amount of enterovirus in the substitution typical curve equation 1, get final product the content of enterovirus in the calculation sample in the substitution formula then 2.
Detection method of the present invention quantitatively accurately, increased substantially the sensitivity of the qualitative RT-PCR detection technique of enterovirus in the existing environment water, the linearity range broadness, the precision height has good specificity, does not have cross reaction with other pathogenic micro-organisms common in the environment.
Description of drawings
Fig. 1 is the recombinant plasmid amplification curve of gradient template amount;
Fig. 2 is an enterovirus real-time fluorescence quantitative PCR typical curve.
The present invention is described in further detail below in conjunction with drawings and Examples.
Embodiment
According to technical scheme of the present invention, at first according to the homology of enteroviral rna 5 ' non-coding region conserved sequence, on the basis of existing primer EV1 and EV2, design a pair of enterovirus universal primer EVN again, this enterovirus universal primer EVN comprises upstream primer EVN1 and downstream primer EVN2, wherein, the nucleotides sequence of upstream primer EVN1 is classified as: 5 '-ACTTCGAGAAGCCTAGTACC-3 '; The sequence of downstream primer EVN2 is: 5 '-TAGGATTAGCCGCATTCAG-3 '; Through compare of analysis on the GenBank database, the sequence that has high similarity with this enterovirus universal primer all derives from various enteroviruses, comprise poliovirus, Coxsackie virus, Echo virus, enterovirns type 71, do not find other microorganism sequence (table 1) that similarity is higher.
Table 1: have the sequence of high similarity with the enterovirus universal primer
Below be the specific embodiment that the contriver provides:
1, equipment and reagent
(1) vacuumfilter;
(2) high speed freezing centrifuge, maximum speed of revolution 14000r/min;
(3) centrifuge tube, 50mL, 1.5mL;
(4) ultraviolet spectrophotometer;
(5) ultraviolet visualization instrument;
(6) constant water bath box
(7) real-time fluorescence quantitative PCR instrument;
(8) poliovirus 1 type;
(9) Vero cell;
(10) pMD18-T carrier;
(11) competence intestinal bacteria XL1-blue:
(12) mixed cellulose ester microporous membrane, aperture 0.22 μ m, diameter 50mm;
(13) 2.5mol/L MgCl
2Solution;
(14) 1mol/L hydrochloric acid;
(15) 3% extractum carniss-0.05mol/L glycine buffer (pH=9.5);
(16) polyoxyethylene glycol, molecular weight 6000;
(17) 5mol/L NaCl solution;
(18) IPTG/X-gal/Amp flat board;
(19) LB liquid nutrient medium;
(20) total RNA extraction reagent box;
(21) AMV reversed transcriptive enzyme;
(22) RNA enzyme inhibitors
(23)dNTP?mixture
(24)10×RT?buffer
(25) SYBR Green I real-time fluorescence quantitative PCR test kit;
(26) enterovirus universal primer EV comprises upstream primer EV1 and downstream primer EV2; The nucleotides sequence of upstream primer EV1 is classified as: 5 '-CAAGCACTTCTGTTTCCCCGG-3 ', the nucleotides sequence of downstream primer EV2 is classified as: 5 '-ACCCATAGTAGTCGGTTCCGC-3 '.
(27) enterovirus universal primer EVN comprises upstream primer EVN1 and downstream primer EVN2; The nucleotides sequence of upstream primer EVN1 is classified as: 5 '-ACTTCGAGAAGCCTAGTACC-3 '; The nucleotides sequence of downstream primer EVN2 is classified as: 5 '-TAGGATTAGCCGCATTCAG-3 ', amplified fragments is 231bp.The enterovirus universal primer is synthetic by professional bio-engineering corporation.
2. the preparation of standard substance
Poliovirus is inoculated in the Vero cell, when cell virus reaches 3+～4+, collects sick cell.Extracting the RNA in the sample, is primer with EV2, adds the AMV reversed transcriptive enzyme, carries out reverse transcription according to ordinary method.The reverse transcription product places-20 ℃ of preservations.
With the reverse transcription product is template, and EV1 and EV2 are primer, carry out pcr amplification.The purpose product fragment of gained is reclaimed purifying, be connected with the pMD18-T carrier and be converted among the intestinal bacteria XL1-blue.Select positive bacteria with the IPTG/X-gal/Amp flat screen and fall, in containing the LB liquid nutrient medium of Amp, cultivate, extract plasmid and carry out sequencing.Quantitative with ultraviolet spectrophotometer again ,-20 ℃ of preservations are as the standard substance of fluorescence real-time quantitative PCR behind the gradient dilution.
Utilize 2.31GEC/ μ L～2.31 * 10
9The recombinant plasmid of ten concentration gradients of GEC/ μ L carries out real-time fluorescence quantitative PCR as template according to the step (6) of the program of mensuration, the sample determination of each concentration 5 times, and averaging returns, and obtains the typical curve equation:
C
T=-3.6896logX
0+ 42.987 (R
2=0.997) (formula 1)
In the formula: X
0---original template amount (GEC)
Enterovirus content in the water sample: 40X
0(GEC) (formula 2)
3. mensuration program
(1) sample collecting: at the water sampling 1L of underwater 0.3m place, put into 4 ℃ of ice chests after the water sampling immediately and preserve, detect in the 6h.
(2) the preliminary of virus concentrates: with the mixed cellulose ester filter membrane vacuumfilter of packing into, connect vacuum pump.Water sampling 1L (water sample that suspended particulate is more carries out pre-filtering with gauze earlier) adds 2.5mol/LMgCl
2Solution makes Mg
2+Final concentration is 0.05mol/L, uses the pH=3.5 of 1mol/L hydrochloric acid conditioning solution again, stirring and evenly mixing on magnetic stirring apparatus.Pass through filter membrane with negative pressure leaching.After filtration finishes, filter membrane is transferred in the clean beaker, added extractum carnis-glycine elutriant (pH=9.5), fully soak into filter membrane, magnetic agitation wash-out 30min discards filter membrane then, collects elutriant.
(3) Bing Du secondary concentration: in elutriant, add PEG6000 and 5mol/L NaCl, make final concentration reach 130g/L and 0.2mol/L respectively, stirring and evenly mixing, 4 ℃ of standing over night.In high speed freezing centrifuge, 10000r/min, 4 ℃, centrifugal 30min abandons supernatant liquor, keeps precipitation.
(4) RNA extracts: utilize the total RNA extraction reagent box, according to explanation, extract the RNA in the precipitation, last constant volume is 52 μ L.
(5) reverse transcription: with 13 μ L RNA and 1 μ L, 25 μ mol/L EV2 primer mixings, add the AMV reversed transcriptive enzyme, 70 ℃ of water-bath 10min, ice bath 10min, instantaneous centrifugal after, add 2 μ L, 10 * RTbuffer, 2 μ L dNTP mixture (40mmol/L altogether), 10U RNA enzyme inhibitors and 5U AMV enzyme, 43 ℃ of water-bath 1h, 70 ℃ of water-bath 10min.Instantaneous centrifugal, obtain the reverse transcription product as pcr template.
(6) real-time fluorescence quantitative RT-PCR detects: get 2 * SYBR Mix, 12.5 μ L, each 0.5 μ L of 10 μ mol/LEVN1 and 10 μ mol/L EVN2,2 μ L 25mmol/L MgCl
2, Taq enzyme 2U adds template 2 μ L, complements to 25 μ L with aseptic ultrapure water.Do the negative contrast of template with aseptic ultrapure water.Increase and interpretation of result on the real-time quantitative PCR instrument, reaction conditions is:
1. 94 ℃ of pre-sex change 4min;
2. amplification cycles is 40 times: 94 ℃, and 30s; 55 ℃, 30s; 72 ℃, 30s;
3. collect fluorescent signal at 85 ℃.Carry out the melting curve analysis after the loop ends, be warming up to 95 ℃, continue to detect fluorescence in the whole process from 65 ℃ with 0.2 ℃/s speed.
(7) calculate: according to the C that detects gained
TCalculate the original template amount of enterovirus in the value, substitution typical curve equation (formula 1), get final product the content of enterovirus in the calculation sample in the substitution formula then 2.
Method of the present invention is compared with the qualitative RT-PCR detection technique of enterovirus in the existing environment water, has following significant advantage:
(1) accurately quantitatively:
The present invention adopts the recombinant plasmid by a series of gradient dilutions to carry out real-time fluorescence quantitative PCR as template and makes enterovirus typical curve (Fig. 1,2), can carry out detection by quantitative to the enterovirus content in the water sample according to formula 1 and formula 2.
(2) sensitivity increases substantially
The sensitivity of the qualitative RT-PCR detection technique of enterovirus is 38CCID in the existing environment water
50, and the sensitivity of present method reaches 4.62GEC.(CCID
50(cell cultures 50 3nfective dose and GEC (genome equivalent copy number)) though between do not have definite quantitative relation, CCID
50Unit is much larger than GEC)
(3) linearity range broadness
The linearity range interval of common virus real-time fluorescence quantitative RT-PCR detecting method is 9 orders of magnitude, but present method is worked as the template amount 4.62～4.62 * 10
9In the GEC scope, template amount logarithmic value and C
TBetween have good linear relationship (R
2=0.997).
(4) precision height
Sample C
TThe variation within batch coefficient of value all is lower than 2%, and interassay coefficient of variation all is lower than 5%, illustrates that present method has higher precision (table 2 and table 3).
Table 2: the variation within batch coefficient of test sample
Table 3: the interassay coefficient of variation of test sample
(5) has good specificity
There is not cross reaction with other common in environment pathogenic micro-organisms.
Claims (1)
1. the quantitative detecting method of enterovirus in the environment water is characterized in that, comprises the following steps:
Step 1, enterovirus universal primer EVN design:
Homology according to enteroviral rna 5 ' non-coding region conserved sequence, on the basis of existing primer EV1 and EV2, design a pair of enterovirus universal primer EVN again, this enterovirus universal primer EVN comprises upstream primer EVN1 and downstream primer EVN2, wherein, the nucleotides sequence of upstream primer EVN1 is classified as: 5 '-ACTTCGAGAAGCCTAGTACC-3 '; The sequence of downstream primer EVN2 is: 5 '-TAGGATTAGCCGCATTCAG-3 ';
The nucleotides sequence of described primer EV1 is classified as: 5 '-CAAGCACTTCTGTTTCCCCGG-3 ', the nucleotides sequence of primer EV2 is classified as: 5 '-ACCCATAGTAGTCGGTTCCGC-3 ';
Step 2, the preparation of standard substance:
Poliovirus being inoculated in the Vero cell, when cell virus reaches 3+～4+, collecting sick cell, extract the RNA in the sample, is primer with EV2, adds the AMV reversed transcriptive enzyme and carries out reverse transcription, and the reverse transcription product places-20 ℃ of preservations;
With the reverse transcription product is template, EV1 and EV2 are primer, carry out pcr amplification, the purpose product fragment of gained is reclaimed purifying, be connected with the pMD18-T carrier and be converted in the intestinal bacteria, select positive bacteria with flat screen and fall, in containing the LB liquid nutrient medium of Amp, cultivate, extract plasmid and carry out sequencing, quantitative with ultraviolet spectrophotometer again ,-20 ℃ of preservations are as the standard substance of fluorescence real-time quantitative PCR behind the gradient dilution;
Utilize 2.31GEC/ μ L～2.31 * 10
9The recombinant plasmid of ten concentration gradients of GEC/ μ L is as template, and wherein, GEC is the genome equivalent copy number, carries out real-time fluorescence quantitative PCR according to the mensuration program, the sample determination of each concentration 5 times, and averaging returns, and obtains the typical curve equation:
C
T=-3.6896logX
0+ 42.987R
2=0.997 formula 1
In the formula: X
0---original template amount GEC
Enterovirus content in the water sample: 40X
0GEC formula 2
Step 3, detection by quantitative:
1) sample collecting:, put into 4 ℃ of ice chests after the water sampling and preserve at underwater 0.3m place water sampling;
2) the preliminary of virus concentrates: with the mixed cellulose ester filter membrane vacuumfilter of packing into, filter the water sample that contains enterovirus, carry out membrane elution with extractum carnis-glycine buffer as eluent, the collection elutriant;
3) secondary concentration: add PEG and NaCl in elutriant, 4 ℃ of standing over night stirring and evenly mixings staticly settle under 4 ℃ and spend the night, and are centrifugal, collecting precipitation;
4) RNA extracts: utilize RNA to extract test kit, extract viral RNA from precipitation;
5) reverse transcription: with 13 μ L RNA and 1 μ L, 25 μ mol/L EV2 primer mixings, add the AMV reversed transcriptive enzyme, 70 ℃ of water-bath 10min, ice bath 10min, instantaneous centrifugal after, add 2 μ L, 10 * RTbuffer, 2 μ L dNTP mixture are 40mmol/L altogether, 10U RNA enzyme inhibitors and 5U AMV enzyme, 43 ℃ of water-bath 1h, 70 ℃ of water-bath 10min, centrifugal, obtain the reverse transcription product as pcr template;
6) real-time fluorescence quantitative RT-PCR detects: get 2 * SYBR Mix, 12.5 μ L, each 0.5 μ L of 10 μ mol/L upstream primer EVN1 and 10 μ mol/L downstream primer EVN2,2 μ L 25mmol/L MgCl
2, Taq enzyme 2U adds template 2 μ L, complements to 25 μ L with aseptic ultrapure water, does the negative contrast of template with aseptic ultrapure water, increases and interpretation of result on the real-time quantitative PCR instrument;
Reaction conditions is:
1. 94 ℃ of pre-sex change 4min;
2. amplification cycles is 40 times: 94 ℃, and 30s; 55 ℃, 30s; 72 ℃, 30s;
3. collect fluorescent signal at 85 ℃, carry out the melting curve analysis after the loop ends, be warming up to 95 ℃, continue to detect fluorescence in the whole process from 65 ℃ with 0.2 ℃/s speed;
7) calculate: according to the C that detects gained
TBe worth, calculate the original template amount of enterovirus in the formula 1 of substitution typical curve equation, get final product the content of enterovirus in the calculation sample in the substitution formula then 2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102888469A (en) * | 2012-10-15 | 2013-01-23 | 西安建筑科技大学 | Method for detecting content of human enteroviruses and non-human enteroviruses in environmental water |
| CN103305630A (en) * | 2013-04-16 | 2013-09-18 | 西安建筑科技大学 | Detection method for CVA10 in environmental water body |
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| CN102021248B (en) * | 2009-09-09 | 2012-12-26 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Method and kit for detecting poliomyelitis viruses in food in real time through fluorescent RT-PCR |
| CN103305628B (en) * | 2013-04-16 | 2015-07-15 | 西安建筑科技大学 | Detection method for EV71virus in environmental water body |
| CN103305629B (en) * | 2013-04-16 | 2015-07-15 | 西安建筑科技大学 | Detection method for CVA16 in environmental water body |
| CN103760122A (en) * | 2014-01-03 | 2014-04-30 | 深圳市宇驰检测技术有限公司 | Ultraviolet visible spectrophotometric method of escherichia coli |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102888469A (en) * | 2012-10-15 | 2013-01-23 | 西安建筑科技大学 | Method for detecting content of human enteroviruses and non-human enteroviruses in environmental water |
| CN103305630A (en) * | 2013-04-16 | 2013-09-18 | 西安建筑科技大学 | Detection method for CVA10 in environmental water body |
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