CN102634607A - H9 subtype avian influenza virus loop-mediated isothermal amplification micro total analysis method - Google Patents
H9 subtype avian influenza virus loop-mediated isothermal amplification micro total analysis method Download PDFInfo
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Abstract
The invention provides an H9 subtype avian influenza virus loop-mediated isothermal amplification micro total analysis method, wherein automatic pretreatment and nucleic acid loop-mediated isothermal amplification are performed on the H9 subtype avian influenza virus on a micro total analysis system (figure 1) driven by a micro diaphragm pump (valve). The method comprises the following steps of: 1) virus splitting; 2) nucleic acid extraction and purification; and 3) nucleic acid amplification. The steps of virus splitting, nucleic acid extraction and purification, nucleic acid amplification and the like are all integrated on a micro total analysis system and automatically finished. The result can be judged by use of a direct fluorescence visual method, an ultraviolet irradiation fluorescence visual method, a gel electrophoresis method and the like. Compared with the traditional RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method, the detection sensitivity of the method can be improved by about 102 times, and the detection time is shortened from 4-5 hours to about 1 hour.
Description
Technical field
The present invention relates to a kind of detection method of virus antigen, relate in particular to a kind of detection H9 subtype avian influenza virus ring mediated isothermal amplification micro-total analysis method.
Background technology
(Avian influenza is a kind of acute, the height contagious disease that is caused by AI virus (AIV) AI), only infects birds usually, and rare situation can infected pigs in bird flu.According to virus surface structural protein hemagglutinin (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA) antigenic difference can be divided into 16 H hypotypes and 9 N hypotypes with bird flu virus.Although the H9 subtype avian influenza be the low pathogenicity bird flu (Lowly pathogenic avian influenza, LPAI), it is propagated extensively, can cause the humans and animals morbidity, even causes the outburst of disease and popular, harm can not be ignored.Therefore, the H9 subtype avian influenza virus detection technique of easy, quick, highly sensitive, high specific has the instant current demand.
Existing avian flu virus detection method mainly is divided into antibody test and antigen detection method.Antibody detection method comprises: hemagglutination-inhibition test (Hemagglutination inhibition; HI), agar immunodiffusion (Agarose gel immunodiffusion; AGID), EUSA (Enzyme-linked Immunosorbent Assay; ELISA), complement fixation test (CFT) (Complement FixationAssays; CF), the neuraminidase inhibition test (Neuraminidase inhibitor test, NIT) and virus neutralization tests (Virusneutralization Test, VNT) etc.Antibody detection method is not because of distinguishing manual injection's vaccine and virus infection, so using value is little in reality, so be the main flow detection method of AIV based on the Detection of antigen technology at present.
To the AIV Detection of antigen, classical gold standard detection method is a chicken embryo virus isolation identification technology, and this method sense cycle is long, and operation steps is numerous and diverse.Common method for quick comprises ELISA and fluorescence RT-PCR.The two is compared, and the ELISA detection sensitivity is lower, immunoreagent quality stability, batch between repeatability relatively poor; And RT-PCR technology susceptibility is high, specificity is good, is just more and more accepted by people.Yet traditional RT-PCR method needs through " high-temperature denatured, middle temperature extension, low-temperature annealing " equitemperature working cycle on the one hand; On the other hand; Steps such as the related lysis of sample preparation, nucleic acid extraction purifying, amplification need use several (cover) equipment to realize at the several separate detection zone; Therefore have following defective in the practical application: it is higher that cost is detected in (1), and grass-roots unit is difficult to use; (2) can not in plant, quaratine building and clinical etc. the place be on-the-spot uses, must be equipped with special laboratory, need to use special-purpose PCR appearance with thermal cycling function; (3) complex operation, has directly influenced the discovery early and the prevention and control of epidemic situation at detection time tediously long (more than several hours).
Get into 21 century; Japan scientist Notomi etc. develops ring mediated isothermal amplification (loop-mediated isothermalamplification; LAMP) technology, nucleic acid chains realizes the amplification to target sequence through the amplification of the cyclic permutation in isothermal environment, does not need the PCR appearance.The amplification efficiency of LAMP is at present the highest a kind of, and its remolding sensitivity conventional P CR exceeds about 2-3 one magnitude.The DNA quantity of amplified production is high more a lot of than PCR product, can reach several micrograms.Yet, because this technology huge in sensitive and product amount too makes LAMP contaminated extremely easily.The LAMP product of 1 amplification often is again same segmental Tumor-necrosis factor glycoproteins, and therefore, if in a single day the laboratory suffers that aerosol pollutes, false positive rate can be very high and be difficult to remove.So the someone thinks, the application direction that LAMP is final should be covered detection.The Manz etc. of Switzerland proposed micro-total analysis (notion of μ-TAS) also was referred to as chip lab (Lab on a Chip) or micro-fluidic chip (Microfluidic Chip) technology, has advantages such as robotization, integrated, portability nineteen ninety.
Therefore; LAMP and μ-TAS are carried out the system integration; Utilize the two unique technique advantage separately, exactly can address the above problem, provide a kind of easy, quick, susceptibility is high, high specificity, bird flu virus nucleic acid molecule detection method that level of automation is high; On micro-total analysis system, realize lysis, nucleic acid extraction purifying, nucleic acid amplification and the detection of sample, total analysis is the sequencing automatic control process.
Summary of the invention
The purpose of this invention is to provide a kind of detection H9 subtype avian influenza virus ring mediated isothermal amplification micro-total analysis method.The present invention uses loop-mediated isothermal amplification technique on micro-total analysis system; Can realize comprising the total analysis process of sample process; Like whole robotizations of steps such as lysis, nucleic acid extraction purifying, isothermal duplication, have the susceptibility height, specificity is good and characteristics such as quick, and cheap; Be applicable to the field quick detection bird flu virus, thus overcome present method for nucleic acid analysis trivial operations, time-consuming, be easy to pollute and must be in problems such as several separate subregion laboratory completion.
The purpose of this invention is to provide secretory product, serum, urine, tracheae examination that a kind of robotization, quick, easy, sensitive and economic being used for detect the human or animal, let out abdominal cavity examination, ight soil or organize the nucleic acid molecule isothermal duplication micro-total analysis method of vat liquor bird flu virus.The present invention utilizes the micro-total analysis system of automatic routine control to realize loop-mediated isothermal amplification technique; From the sample to be tested of living, extract nucleic acid; Utilize institute's designed primer to carry out amplified reaction; Adopt three kinds of detection modes: whether (one) is the fluorescence visual method directly, under visible light, have tangible fluorescence to judge whether sample to be tested contains bird flu virus in the direct viewing reaction tubes; (2) UV-irradiation fluorescence visual method, the fluorescence of observing response pipe judges whether sample to be tested contains bird flu virus under ultraviolet excitation; Whether (three) gel electrophoresis, detection reaction product contain specific scalariform band.
The present invention tries son, lets out abdominal cavity examination, ight soil or organize vat liquor amplifying nucleic acid extract with human or animal's secretory product, serum, urine, tracheae is template; One section high conservative fragment selecting H9 subtype avian influenza virus HA gene is as target sequence, employed specificity 6 Auele Specific Primers such as tables 1.Wherein, LF and LB are respectively between F1C and F2, the ring primer between BIC and the B2, and its effect is to improve amplification efficiency, accelerates speed of reaction.
Table 1 H9 subtype avian influenza virus RT-LAMP primer sequence
H9 subtype avian influenza virus ring mediated isothermal amplification micro-total analysis method provided by the invention; Lysis, nucleic acid extraction purifying, isothermal duplication with sample to be tested; All be integrated on the micro-total analysis system (see figure 1) and accomplish automatically; This micro-total analysis system is driven by sheet pump (valve), through the unlatching and the closed drive controlling that realizes microfluid sample of time variable control sheet pump (valve).Whole analytical procedure is following:
1. nucleic acid extraction purifying
1) lysis
Get the sample inlet pool (1) that the sample to be tested 5-50 μ L that contains H9 subtype avian influenza virus RNA template is injected into the micro-total analysis system that is driven by mocromembrane pump (valve); Get cell pyrolysis liquid 20-50 μ L and be injected into extraction reagent sample holes (2); Lysate flows into sample inlet pool (1), cracking 10min under the room temperature condition by extracting reagent sample holes (2) under the mocromembrane pump drives.To be injected into the isopyknic ethanol of lysate and extract reagent sample holes (2), drive inflow sample inlet pool (1) down, with cracked sample mixed at the mocromembrane pump.
2) nucleic acid extraction and wash-out
Above-mentioned mixed solution flow into purifying hole (4) by sample inlet pool (1) under the mocromembrane pump drives; Through the plain film enrichment of silicate fiber; Flow into waste liquid pool (3); Get 50-100 μ L leacheate and join extraction reagent sample holes (2), drive dirty purified hole (4) at membrane pump the silicic acid film is washed back inflow waste liquid pool (3), carry out wash-out with the above-mentioned washing operation of 50-100 μ L elutriant.
2. nucleic acid amplification
Get with the isopyknic extracting solution of sample and be injected into amplifing reagent sample holes (5); After under membrane pump drives, flowing into purifying hole (4) and the purified RNA template mixed; Flow into again in the LAMP reaction tubes and mix, in 60-65 ℃ of following isothermal duplication 15-50min with the 10 μ L amplification reaction solutions that preset.
Amplification reaction system is: 10 * ThermoPoL Buffer, 2.5 μ L, 8U/ μ L Bst DNA Polymerase 1 μ L, 5U/ μ LAMV reverse transcriptase 1 μ L; 10mmol/L dNTPs 1 μ L; 2-10mmol/L trimethyl-glycine 5 μ L, 50-150mmol/LMgSO4 1 μ L, each 1 μ L of 40 μ mol/L FIP/BIP, each 0.5 μ L of 10 μ mol/L F3/B3, each 1 μ L of 20 μ mol/L LF/LB; RNA template 5 μ L complement to 25 μ L with DEPC water.Replace the RNA template as negative control with 5 μ LDEPC water.
3. detect
1) direct fluorescence visual method
Add 1 μ L fluorexon in the amplified reaction forward direction amplification reaction system as fluorescent indicator, directly through naked-eye observation, the positive result of greeny sample is the negative result of more shallow orange to reaction result under visible light.
2) UV-irradiation fluorescence visual method
Add 1 μ L fluorexon in the amplified reaction forward direction amplification reaction system as fluorescent indicator, irradiation under UV-light, the positive result of the fluorescence that takes on a red color, the negative result who does not develop the color.
3) gel electrophoresis
Get amplified production 3 μ L, adopt 1% agarose gel electrophoresis method to detect, the positive result of obvious trapezoid-shaped strips person is arranged; Otherwise negative result.
Method of the present invention is when concrete the application; Wherein said testing sample is extracting solutions such as human or animal's secretory product, serum, urine or tissue, internal organs; The ring mediated isothermal amplification that on micro-total analysis system, is carried out, the suitable time of amplified reaction are 15-50min, and the preferred time is 30min; The optimal temperature of amplified reaction is 60-65 ℃, and preferred temperature is 63 ℃; The employed trimethyl-glycine concentration of amplified reaction is 2-10mmol/L, and preferred concentration is 5mmol/L; The employed MgSO4 strength of solution of amplified reaction is 50-150mmol/L, and preferred concentration is 100mmol/L.
The present invention compares with traditional RT-PCR method, and sensitivity can improve about 10
2Doubly, foreshortened to about 1 hour detection time from 4-5 hour, and the whole analysis operations that comprise sample preparation all are integrated on the micro-total analysis system and carry out, and through time variable control, robotization is accomplished.
Research unit, company that H9 subtype avian influenza antigen required for the present invention can arrive relevant speciality buy or customization; Required instrument, equipment, reagent all have commercially available.
Description of drawings
Fig. 1: micro-total analysis system structural representation.
Fig. 2: the optimization experiment in H9-RT-LAMP reaction times is figure as a result.
1～5 is respectively the H9-AIV RNA of reaction times 20～60min, 6 negative contrasts
Fig. 3: the optimization experiment of H9-RT-LAMP temperature of reaction is figure as a result.
1～6 is respectively the H9-AIV RNA of 60～65 ℃ of temperature of reaction, 7 negative contrasts
The optimization experiment of trimethyl-glycine concentration figure as a result in Fig. 4: the H9-RT-LAMP reaction.
1～5 is respectively the H9-AIV RNA of trimethyl-glycine concentration 0,2mmol/L, 5mmol/L, 10mmol/L, 15mmol/L, the optimum result figure of MgSO4 strength of solution in the 6 negative map 5:H9-RT-LAMP reactions.
1～6 is respectively the H9-AIV RNA of MgSO4 concentration 0,10mmol/L, 50mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 7 negative contrasts
Fig. 6 A: gel electrophoresis is investigated RT-LAMP detection sensitivity experimental result picture.
M is Ladder Marker, and 1～8 is 10
0～10
-8The H9-AIV RNA that doubly dilutes, 9 negative contrasts
Fig. 6 B: directly the fluorescence visual method is investigated RT-LAMP detection sensitivity experimental result picture.
1～8 is the H9-AIV RNA that 100～10-8 doubly dilutes
Fig. 6 C: traditional RT-PCR method detection sensitivity experimental result picture.
M is Ladder Marker, and 1～8 is 10
0～10
-8The H9-AIV RNA that doubly dilutes
Fig. 7 A: gel electrophoresis is investigated RT-LAMP specificity experimental result picture.
M is Ladder Marker, and 1～4 is respectively H9-AIV, H5-AIV, H7-AIV, NDV, 5 negative contrasts
Fig. 7 B: directly the fluorescence visual method is investigated RT-LAMP specificity experimental result picture.
1～4 is respectively H9-AIV, H5-AIV, H7-AIV, NDV, negative control
Embodiment
The present invention does further to specifically describe through following examples.
Embodiment 1: micro-total analysis RT-LAMP reaction times optimization experiment
By above experimental procedure; Other condition inconvenience, setting gradually the micro-total analysis isothermal amplification time is 20min, 30min, 40min, 50min, 60min, experimental result is as shown in Figure 2; The suitable time of judging amplified reaction in view of the above is 20-50min, and the preferred time is 30min.
Embodiment 2: micro-total analysis RT-LAMP temperature of reaction optimization experiment
By above experimental procedure; Other condition inconvenience, setting gradually micro-total analysis isothermal amplification temperature is 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, experimental result is as shown in Figure 3; The optimal temperature of judging amplified reaction in view of the above is 60-65 ℃, and preferred temperature is 63 ℃.
Embodiment 3: trimethyl-glycine concentration optimization experiment in the micro-total analysis RT-LAMP reaction
By above experimental procedure; The inconvenience of other conditions, trimethyl-glycine concentration are followed successively by 0,2mmol/L, 5mmol/L, 10mmol/L, 15mmol/L, and experimental result is as shown in Figure 4; The trimethyl-glycine concentration of judging amplified reaction in view of the above is 2-10mmol/L for trimethyl-glycine concentration, and preferred concentration is 5mmol/L.
Embodiment 4: MgSO4 strength of solution optimization experiment in the micro-total analysis RT-LAMP reaction
By above experimental procedure, other conditions inconvenience, the MgSO4 strength of solution is followed successively by 0,10mmol/L, 50mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, and experimental result is as shown in Figure 5, judges the MgSO of amplified reaction in view of the above
4Strength of solution is 50-150mmol/L, and preferred concentration is 100mmol/L.
Embodiment 5: the experiment of micro-total analysis RT-LAMP detection sensitivity
The experiment of micro-total analysis RT-LAMP detection sensitivity is carried out series 10 * doubling dilution to the RNA template of H9-AIV; Minimum extension rate is to 10-7; Measure the RNA concentration of diluting preceding each gradient with ultraviolet spectrophotometer, calculate the RNA quality and be respectively 100n g, 10n g, 1ng, 0.1ng, 10pg, 1pg, 100fg, 10fg.Get the RNA 5 μ LRT-LAMP reaction of each extension rate respectively, confirm the sensitivity of this method.Outer primer F3 and B3 with RT-LAMP carries out micro-total analysis RT-PCR reaction as the upstream and downstream primer simultaneously.The relatively sensitivity of two kinds of methods.Shown in Fig. 6 A, 6B; The H9-RT-LAMP method detection sensitivity of being set up is the 10-6 extension rate; Can detect the RNA of 100fg, visual result of determination under the visible light (positive pipe solution is green, and negative tube solution is more shallow orange) is consistent with the agarose gel electrophoresis result.With the amplification of 1% agarose gel electrophoresis judgement RT-PCR, shown in Fig. 6 C, the sensitivity of RT-PCR is the 10-4 extension rate, promptly detects the RNA of 10pg.It is thus clear that the sensitivity of RT-LAMP is 100 times of RT-PCR.
Embodiment 6: the specificity experiment of micro-total analysis RT-LAMP
Shown in Fig. 7 A and 7B, have only H9 hypotype AIV the characteristic trapezoid-shaped strips to occur, other viruses all increase less than band, and visual result of determination under the visible light (positive pipe solution is green, and negative tube solution is more shallow orange) is consistent with the agarose gel electrophoresis result.Show that the H9-RT-LAMP primer that the present invention is directed to the design of H9 subtype avian influenza virus has excellent specificity.
Claims (7)
1.H9 6 Auele Specific Primers of the employed specificity of subtype avian influenza virus ring mediated isothermal amplification micro-total analysis method, LF and LB are respectively between F1C and F2, the ring primer between BIC and the B2, and its primer sequence is:
2. H9 subtype avian influenza virus ring mediated isothermal amplification micro-total analysis method according to claim 1; It is characterized in that; Human or animal's to be measured secretory product, serum, urine, tracheae examination, let out abdominal cavity examination, ight soil or organize pretreatnlent of sample such as vat liquor; Comprise that lysis, nucleic acid extraction purifying, isothermal duplication all are integrated in completion automatically on the micro-total analysis system (see figure 1); This micro-total analysis system is driven by sheet pump (valve), through the unlatching and the closed drive controlling that realizes microfluid sample of time variable control sheet pump (valve).
3. like the said H9 subtype avian influenza virus of claim 2 ring mediated isothermal amplification micro-total analysis method; It is characterized in that; Detection method comprises: 1) direct fluorescence visual method, in amplified reaction forward direction system, add 1 μ L fluorexon as fluorescent indicator, and can be directly through the naked-eye observation result; Greeny positive result is the negative result of more shallow orange; 2) UV-irradiation fluorescence visual method, under under UV-light, shining, the positive result of the fluorescence that takes on a red color, the negative result who does not develop the color; 3) gel electrophoresis has the positive result of obvious trapezoid-shaped strips person, on the contrary negative result.
4. like the said H9 subtype avian influenza virus of claim 3 ring mediated isothermal amplification micro-total analysis method, it is characterized in that the suitable time of amplified reaction is 15-50min, the preferred time is 30min.
5. like the said H9 subtype avian influenza virus of claim 4 ring mediated isothermal amplification micro-total analysis method, it is characterized in that the optimal temperature of amplified reaction is 60-65 ℃, preferred temperature is 63 ℃.
6. like the said H9 subtype avian influenza virus of claim 5 ring mediated isothermal amplification micro-total analysis method, it is characterized in that the employed trimethyl-glycine concentration of amplified reaction is 2-10mmol/L, preferred concentration is 5mmol/L.
7. like the said H9 subtype avian influenza virus of claim 6 ring mediated isothermal amplification micro-total analysis method, it is characterized in that the employed MgSO of amplified reaction
4Strength of solution is 50-150mmol/L, and preferred concentration is 100mmol/L.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575649A (en) * | 2008-06-02 | 2009-11-11 | 中国农业科学院兰州兽医研究所 | Quick detection technology for H9 type avian influenza virus |
| CN102199531A (en) * | 2011-03-30 | 2011-09-28 | 复旦大学 | Microfluidic chip for multiple loop-mediated isothermal amplification (LAMP) detection and preparation method thereof |
| CN102329891A (en) * | 2011-08-31 | 2012-01-25 | 中国农业科学院哈尔滨兽医研究所 | RT-LAMP (reverse transcription-loop-mediated isothermal amplification) primer group for detecting H9 subtype avian influenza virus as well as detection method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575649A (en) * | 2008-06-02 | 2009-11-11 | 中国农业科学院兰州兽医研究所 | Quick detection technology for H9 type avian influenza virus |
| CN102199531A (en) * | 2011-03-30 | 2011-09-28 | 复旦大学 | Microfluidic chip for multiple loop-mediated isothermal amplification (LAMP) detection and preparation method thereof |
| CN102329891A (en) * | 2011-08-31 | 2012-01-25 | 中国农业科学院哈尔滨兽医研究所 | RT-LAMP (reverse transcription-loop-mediated isothermal amplification) primer group for detecting H9 subtype avian influenza virus as well as detection method and application thereof |
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