CN105779656B - Porcine torque teno virus type 2 loop-mediated isothermal amplification kit and application thereof - Google Patents
Porcine torque teno virus type 2 loop-mediated isothermal amplification kit and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a porcine torque teno virus type 2 loop-mediated isothermal amplification kit and application thereof, wherein the kit comprises LAMP primers, 2 multiplied reaction buffer solution, Bst DNA polymerase, fluorescent visual detection reagent, ultrapure water and porcine torque teno virus type 2 DNA template, and the LAMP primers comprise outer primers F3 and B3, inner primers FIP and BIP and loop primers LF and LB. The application of the kit comprises the detection of a porcine torque teno virus type 2 lesion tissue sample and the qualitative research of porcine torque teno virus type 2 pathogeny. The LAMP detection method provided by the invention can monitor the reaction in real time and quantitatively detect the copy number of the porcine torque teno virus type 2, quickly and accurately obtain the detection result, and is convenient for simply, quickly and reliably detecting the porcine torque teno virus type 2.
Description
Technical Field
The invention relates to the technical field of microbial detection, in particular to a loop-mediated isothermal amplification kit capable of rapidly, visually and quantitatively detecting porcine torque teno virus type 2 in real time and application thereof.
Background
Porcine Torque teno virus (TTSuV) is a membrane-free, single-stranded circular DNA virus that, based on its nucleotide sequence differences, divides TTSuV into two subtypes, TTSuV1 and TTSuV2, wherein TTSuV1 is classified as the genus Iota-torquevirus and TTSuV2 is classified as the genus kappaorrque-virus. The minicirus is first discovered in serum of post-transfusion hepatitis patients in 1997 by Nishizawa, a Japanese scholarly, and has a very broad host range, and the virus has now widely infected non-human primates, domestic and wild animals, and humans. The infection rate of the torque teno virus in people is high, and according to epidemiological investigation of TTV infection of different people from different countries, the TTV DNA positive rate of people is generally over 10 percent. In the field of porcine torque teno virus, porcine torque teno virus (TTSuV) is also widely present in swine herds in many countries such as japan, usa, thailand, korea, france, spain, canada, etc., and the infection rate is as high as 24% to 100%. The homology of nucleic acid sequences among TTSuV strains in different regions can reach 86-100%. Because TTSuV2 is widely present in swine herds, its specific biological significance has not been clarified. The pathogenicity of TTSuV2 is unknown so far, and only the research at present knows that TTSuV2 has certain synergistic effect with viruses such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus (PCV), and the symptoms of weaning Pig Multisystemic Wasting Syndrome (PMWS) and porcine dermatitis nephrotic syndrome are aggravated when mixed infection occurs.
Clinical data show that the TTSuV2 content is related to the severity of diseases such as porcine circovirus type 2 (PCV 2), and the like, so that the determination of the viral load of TTSuV2 is of great significance. Because of the lack of commercial reagents for serological detection, conventional PCR, fluorescent quantitative PCR method, nested PCR and other technical means are mainly used for detecting TTSuV 2. However, these techniques have some disadvantages in application and have high requirements on hardware, agarose gel electrophoresis is also needed in result determination, which easily causes laboratory pollution to cause false positive results, and the techniques are long in use time and not suitable for basic level and field detection. Therefore, a simple and sensitive TTSuV2 detection method is established, and has certain clinical significance.
Disclosure of Invention
The invention aims to provide a method for simply, conveniently, quickly and accurately detecting porcine torque teno virus type 2 for a basic layer, and discloses a quick real-time quantitative porcine torque teno virus type 2 loop-mediated isothermal amplification kit. The technical scheme used for realizing the purpose of the invention is as follows: a porcine torque teno virus type 2 loop-mediated isothermal amplification kit comprises LAMP primers, 2 x reaction buffer solution, Bst DNA polymerase, fluorescent visual detection reagent, ultrapure water and a porcine torque teno virus type 2 DNA template, wherein the LAMP primers comprise outer primers F3 (SEQ ID NO: 1) and B3 (SEQ ID NO: 2), inner primers FIP (SEQ ID NO: 3) and BIP (SEQ ID NO: 4) and loop primers LF (SEQ ID NO: 5) and LB (SEQ ID NO: 6);
wherein the sequences of the primers are respectively as follows:
F3 AAGTGCGCAGACGAATGG
B3 GGGTTTTTACAGCCGCAGAA
FIP ACTCCGCTCTCAGGAGCTCCTTTATGCCGCTGGTGGTAG
BIP GCGGTAATCCAGCGGAACCG TAATCCGTGCGCGCAGTA
LF ACACTCAGCTCTGTTCGTGT
LB CCCCCCCTCCATGGAAGAA
the 2 × reaction buffer solution comprises Tris-HCL, KCL and MgSO4、(NH4)2SO4Tween20, Betaine and dNTPs。
The porcine torque teno virus type 2 DNA template is porcine torque teno virus type 2 genome DNA extracted from suspected porcine torque teno virus type 2 lesion tissues or porcine torque teno virus type 2 cultures by using a virus genome DNA/RNA extraction kit.
The fluorescence visual detection reagent adopts calcein fluorescent reagent which is added before reaction.
The 2 × reaction buffer solution comprises 35-55mM Tris-HCL, 10-30mM KCL and MgSO4 15-28mM、(NH4)2SO418-28mM, Twen200.5-1.5% heavy, Betaine 1.3-3.5M and dNTPs 2.4-4.8mM, and the preparation method is to mix the above solvents uniformly under the condition that the pH is about 8.6.
The application of the porcine torque teno virus type 2 loop-mediated isothermal amplification kit is used for detecting whether the genome DNA in clinical lesion tissues or cultures suspected to be infected with the porcine torque teno virus type 2 contains the porcine torque teno virus type 2, and the specific detection steps comprise:
(1) design and synthesis of LAMP primers
(2) Extraction of porcine torque teno virus type 2 DNA template
(3) Establishment of LAMP reaction System
(4) LAMP detection method.
The LAMP reaction system is established by 25 mu L,
2 × reaction buffer 12.5 μ L
Bst DNA polymerase 1. mu.L
FIP 40 pmol
BIP 40 pmol
F3 5 pmol
B3 5 pmol
LF 20 pmol
LP 20 pmol
2 mu L of porcine torque teno virus type 2 DNA
Make up 25. mu.L of ultrapure water.
The LAMP detection method adopts a specific detection method, a sensitivity detection method and a fluorescence visualization detection method.
The LAMP detection method adopts a Loopamp LA-320C real-time turbidimeter to carry out closed whole-process monitoring, the reaction temperature is 63 ℃, and amplification occurs in the reaction within 18-25 minutes.
The substantive features and the remarkable progress of the invention are as follows:
1) strong specificity
The LAMP method of the invention specifically detects the porcine torque teno virus type 2, and the detected negative control virus and water control have no positive result and are consistent with the PCR detection result. And the method is simple and convenient to operate, can quickly obtain the detection result, and does not need expensive and complicated instruments.
2) High sensitivity
The sensitivity of the common PCR detection method is 1.40X 10-7ng/. mu.L, and using the LAMP detection method of the present invention, the detection limit is about 1.40X 10-8ng/. mu.L, 10 times that of ordinary PCR.
3) Obtain results quickly
The result can be obtained only in about 24 hours in the whole process of common PCR, the result is judged by adopting agarose gel electrophoresis ultraviolet development analysis after the reaction of most established LAMP reaction methods at present, and about 4-5 hours is needed for extracting the test result from genome DNA. The LAMP detection method provided by the invention has the advantages that the amplification occurs in about 18 minutes, the amplification can be completed within 60 minutes, the result interpretation mode is simple and convenient, the positive tubes and the negative tubes are compared under visible light, the positive reaction is obviously turbid by naked eyes, and the negative reaction tubes are transparent; after short-time centrifugation, the bottom of the positive reaction tube has obvious white magnesium pyrophosphate precipitate, and the bottom of the negative reaction tube has no precipitate; or fluorescent dye is added, the positive reaction tube is green under ultraviolet light, and the experimental result can be observed by naked eyes. The result is judged without agarose gel electrophoresis ultraviolet analysis and development or uncapping and adding fluorescent dye, and the extraction from the genome DNA and the final result can be completed within 2-3 hours.
4) Does not cause pollution
The fluorescent dye used for direct observation in the LAMP method is added after reaction, while the fluorescent dye is a calcein commercial dye (non-syber-green) added before reaction, and the detection process does not need to open the cover. In addition, in the LAMP detection method, the result is judged by directly detecting the turbidity value of the reaction tube through the turbidity meter on the result judgment, the detection result of a fluorescent dye method or the detection result of agarose gel electrophoresis is not needed, the cover opening is not needed, and the pollution can be effectively avoided.
5) Can be quantified in real time
The LAMP reaction result is analyzed in real time by using a Tubidimeter real-time LA-320 turbidimeter, the time of turbidity values corresponding to different concentrations of standard samples is drawn into a standard curve, and the standard curve is substituted into a standard curve equation, so that the porcine torque teno virus type 2 copy number of each time can be calculated, and the aim of quantitatively detecting products is fulfilled.
Drawings
FIG. 1 shows the result of detection of the specificity of the LAMP method of the present invention; wherein 1: porcine torque teno virus type 2; 2: porcine torque teno virus type 1; 3: porcine circovirus type ii; 4: hog cholera virus; 5: a blue ear disease virus; 6: porcine parvovirus; 7: porcine pseudorabies virus; 8: epidemic diarrhea virus; 9: blank control (water). The rising curve of the turbidity appears in the porcine torque teno virus type 2 reaction tube, which is a positive result, and the 7 strains of the control virus reaction tube and the water control reaction tube have no amplification, which is a negative result.
FIGS. 2 and 3 are results of sensitivity detection of porcine torque teno virus type 2 using the LAMP method of the present invention and the general PCR method, respectively, in which 1: 1.40X 102 ng/μL；2：1.40×10 ng/μL；3：1.40 ng/μL；4：1.40×10- 1ng/μL； 5：1.40×10-2ng/μL；6：1.40×10-3ng/μL；7：1.40×10-4ng/μL；8：1.40×10-5ng/μL；9：1.40×10-6ng/μL；10： 1.40×10-7 ng/μL；11：1.40×10-8ng/mu L; 12: water. Porcine torque teno virus type 2The initial concentration of the original DNA was 1.40X 102ng/mu L, performing LAMP and PCR amplification after 10-fold-ratio continuous dilution, and the result shows that the detection limit of the LAMP method is about 1.40 multiplied by 10-8ng/. mu.L, and the detection limit of the common PCR method is about 1.40X 10-7ng/μL。
FIG. 4 is a visual inspection of the sample after addition of the fluorescent dye: the left tube shows the positive result when the porcine torque teno virus type 2 genomic DNA is used as a template, and the right tube shows the negative result when the right tube shows the negative control.
FIG. 5 is a quantitative standard curve of porcine torque teno virus type 2 of the present invention; and (3) drawing a standard curve by using turbidity values corresponding to the concentrations of different standard samples to time, and substituting the standard curve equation to obtain the porcine torque teno virus type 2 copy number of each time.
Detailed Description
1. Preparation of the Material
Porcine torque teno virus type 2, porcine torque teno virus type 1, porcine torque teno virus type 2, hog cholera virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine pseudorabies virus and epidemic diarrhea virus; isolated, identified and stored for commercial vaccine virus or Guangxi veterinary institute. The LAMP DNA amplification kit is purchased from Beijing blue Spectrum Biotechnology Ltd, the product number is LMP 204; the viral genome DNA/RNA extraction kit was purchased from Shikoku Biotechnology Ltd, cat # CW 0552.
2. Design and synthesis of LAMP primers
According to the gene sequence of ORF1 of porcine torque teno virus type 2 in GenBank, LAMP primers are designed by using primer auxiliary design software PrimeExplorer V4 software of LAMP method, wherein F3 and B3 are outer primers, FIP and BIP are inner primers, and LF and LB are loop primers; wherein F3 and B3 are porcine torque teno virus type 2 PCR detection primers, wherein
F3 AAGTGCGCAGACGAATGG
B3 GGGTTTTTACAGCCGCAGAA
FIP ACTCCGCTCTCAGGAGCTCCTTTATGCCGCTGGTGGTAG
BIP GCGGTAATCCAGCGGAACCG TAATCCGTGCGCGCAGTA
LF ACACTCAGCTCTGTTCGTGT
LB CCCCCCCTCCATGGAAGAA。
3. Viral genomic DNA extraction
The viral genomic DNA/RNA extraction kit produced by Hakken Biotech Co., Ltd was used to extract the porcine torque teno virus type 2 DNA or the genomic DNA of a lesion tissue suspected of being infected with porcine torque teno virus type 2, and the genomic DNA of a control virus.
4. Establishment of LAMP reaction System
According to the kit instruction, the preparation is carried out according to a 25-mu-L system:
2 × reaction buffer 12.5 μ L
Bst DNA polymerase 1. mu.L
FIP 40 pmol
BIP 40 pmol
F3 5 pmol
B3 5 pmol
LF 20 pmol
LP 20 pmol
2 mu L of porcine torque teno virus type 2 DNA
Make up 25. mu.L of ultrapure water.
The LAMP reaction is monitored in a mode of closed whole-process monitoring by a real-time turbidimeter (LA-320C, Japan research and development company), the amplification condition is monitored by the turbidimeter in real time, a standard curve can be drawn, the initial copy number of an unknown sample can be calculated from the standard curve by obtaining a time value corresponding to the turbidity value of the unknown sample reaching 0.1, and the reaction temperature is 63 ℃ as the reaction temperature.
5. LAMP detection method
1) Specificity detection
The virus genome DNA/RNA extraction kit is used for extracting the genome DNA of porcine torque teno virus type 2, porcine torque teno virus type 1, porcine torque teno virus type II, hog cholera virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine pseudorabies virus and epidemic diarrhea virus, and the genome DNA is used as a template of LAMP reaction to carry out LAMP amplification, and simultaneously water is used as blank control to test the specificity of the LAMP method.
2) Sensitivity detection
Extracting porcine torque teno virus type 2 genome DNA, measuring the concentration of the extracted porcine torque teno virus type 2 genome DNA, continuously diluting the extracted porcine torque teno virus type 2 genome DNA into 11 dilutions by using an RNA-Free Water 10-fold ratio, carrying out LAMP method amplification and conventional PCR amplification by using each DNA dilution as a template, and comparing the sensitivity of the LAMP method and the common PCR to the detection of the porcine torque teno virus type 2.
3) Fluorescence visualization detection
According to optimized conditions monitored and controlled by a turbidimeter, a fluorescent dye is added before reaction, the added dye is a calcein commercial dye, the calcein commercial dye is reacted for 60 minutes at 63 ℃, observation is carried out under an ultraviolet lamp, agarose gel electrophoresis ultraviolet analysis development is not adopted, and aerosol pollution to a laboratory caused by uncapping running electrophoresis observation is avoided.
Example 1 specificity results of LAMP detection method
LAMP amplification is carried out on 1 porcine torque teno virus type 2 and 7 control viruses and a water control, the result is shown in figure 1, a turbidity rising curve appears in a porcine torque teno virus type 2 reaction tube in about 18 minutes, the result is a positive result, and the result is a negative result if no amplification condition appears in the 7 control virus reaction tube and the water control reaction tube.
Example 2 sensitivity results of LAMP detection method
The initial concentration of porcine torque teno virus type 2 primary DNA was 1.40X 102ng/. mu.L, 10-fold serial dilution, LAMP and PCR amplification, the results are shown in FIGS. 2 and 3, and the LAMP method of the present invention has a detection limit of about 1.40X 10-8ng/. mu.L, and the detection limit of the conventional PCR method is 1.40X 10-7ng/μL。
Example 3 fluorescence visualization assay results of LAMP detection method
According to optimized conditions monitored by a turbidimeter, fluorescent dye is added into the reactor, the reaction is carried out for 60 minutes at 63 ℃, then the reaction is observed under an ultraviolet lamp, the result is shown in figure 4, the left tube is the reaction condition with the porcine torque teno virus type 2 genome DNA as a template and is a positive result, and the right tube is a negative control and is a negative result. The test result shows that the established LAMP method can be conveniently used in the basic level, the kit is only needed to be matched with the LAMP primer designed by the method, after the sample is added, a cheap water bath kettle is used for keeping the temperature of 63 ℃ for 60 minutes, the result can be quickly observed, the cover is not required to be opened, and the pollution is avoided.
Example 4 plotting of quantitative Standard Curve of porcine torque teno virus type 2
The porcine torque teno virus type 2 DNA is used as a template, outer primers F3 and B3 designed by the LAMP method are used as PCR amplification primers, a target gene segment obtained by PCR amplification is connected to a vector pMD18-T, an escherichia coli sensing cell DH5a is transformed, a monoclonal bacterium is obtained by ampicillin resistance screening, recombinant plasmid DNA is extracted, after sequencing confirmation, the initial concentration of the recombinant plasmid pMD18-T-ORF1 is determined and used as a standard sample, RNA-Free Water is used for carrying out continuous 10-fold dilution with 11 dilutions, and 2 mu L of each dilution is used as a template for LAMP amplification.
Setting comparison: the concentration is 1.4X 102 ng/μL、1.4×101 ng/μL、1.4 ng/μL、1.4×10-1 ng/μL、1.4×10-2 ng/μL、1.4×10-3ng/μL、1.4×10-4 ng/μL、1.4×10-5 ng/μL、1.4×10-6 ng/μL、1.4×10-7 ng/. mu.L and 1.4X 10-8One each for ng/. mu.L of the recombinant plasmid pMD18-T-ORF1 standard sample, since the negative logarithm of the sample concentration is linear with the time value of 0.1 in the amplified turbidity value, the value captured by the nephelometer can be plotted against time (as in Table 1) to obtain a standard curve equation, y = 0.3113x-7.2366, as shown in FIG. 5. The correlation coefficient R is seen from the standard curve equation2 = 0.9951, in good linear relationship. When the time is taken as the X value, the Y value, that is, the negative power of the concentration is obtained, and the concentration is 10-yThen multiplied by the base number 1.4 to obtain 1.4 × 10-yng/. mu.L. Copy number conversion formula copies/. mu.L = (6.02X 10)23 ×(ng/ul ×10-9) /(DNA length. times.660) where 2693+219=2912 bp, which is the vector sequence size plus the target gene sequence size, is converted toCopy number (copies/. mu.L): 6.02X 1023 ×（1.4×10-y × 10-9) (2912 × 660), after simplification: 4.38X 108 × 10-y. If the time for a test sample to reach a turbidity value of 0.1 is 35 minutes, the established standard curve equation is substituted, Y is calculated to be equal to 3.689, and the concentration is 10-3.689And multiplying by the base number 1.4 to obtain the concentration of the test sample of 1.4 multiplied by 10-3.689ng/. mu.L, the copy number is: 4.38X 108 × 10-3.689=4.38 ×104.3411copies/. mu.L, thereby achieving the quantitative effect.
TABLE 1 time-linear relationship between the negative logarithm of the sample concentration and the TTSuV2-LAMP turbidity value
| Time (minutes) | 17.5 | 20 | 23 | 26 | 30 | 32 | 36 | 40 |
| Negative logarithm of concentration | -2 | -1 | 0 | 1 | 2 | 3 | 4 | 5 |
Sequence listing
<110> Guangxi Zhuang nationality autonomous region veterinary research institute
<120> porcine torque teno virus type 2 loop-mediated isothermal amplification kit and application thereof
<160> 6
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence
<400> 1
AAGTGCGCAGACGAATGG
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
GGGTTTTTACAGCCGCAGAA
<210> 3
<211> 39
<212> DNA
<213> Artificial sequence
<400> 3
ACTCCGCTCTCAGGAGCTCCTTTATGCCGCTGGTGGTAG
<210> 4
<211>38
<212> DNA
<213> Artificial sequence
<400> 4
GCGGTAATCCAGCGGAACCG TAATCCGTGCGCGCAGTA
<210> 5
<211>20
<212> DNA
<213> Artificial sequence
<400> 5
ACACTCAGCTCTGTTCGTGT
<210> 6
<211>19
<212> DNA
<213> Artificial sequence
<400> 6
CCCCCCCTCCATGGAAGAA
Claims (1)
1. A porcine torque teno virus type 2 loop-mediated isothermal amplification kit is characterized by comprising LAMP primers, wherein the primers comprise outer primers F3 (SEQ ID NO: 1) and B3 (SEQ ID NO: 2), inner primers FIP (SEQ ID NO: 3) and BIP (SEQ ID NO: 4), loop primers LF (SEQ ID NO: 5) and LB (SEQ ID NO: 6);
wherein the sequences of the primers are respectively as follows:
F3 AAGTGCGCAGACGAATGG
B3 GGGTTTTTACAGCCGCAGAA
FIP ACTCCGCTCTCAGGAGCTCCTTTATGCCGCTGGTGGTAG
BIP GCGGTAATCCAGCGGAACCG TAATCCGTGCGCGCAGTA
LF ACACTCAGCTCTGTTCGTGT
LB CCCCCCCTCCATGGAAGAA；
the kit also comprises 2 multiplied reaction buffer solution, Bst DNA polymerase, fluorescent visual detection reagent, ultrapure water and porcine torque teno virus type 2 DNA template; the 2 × reaction buffer solution comprises Tris-HCL, KCL, MgSO4, (NH4)2SO4, Tween20, Betaine and dNTPs;
the fluorescence visual detection reagent adopts a calcein fluorescent reagent which is added before reaction;
the 2 × reaction buffer solution comprises 35-55mM of Tris-HCL, 10-30mM of KCL, 415-28 mM of MgSO (MgSO 415), 2SO 418-28 mM of (NH4), 200.5-1.5% of Twenn, 1.3-3.5M of Betaine and 2.4-4.8mM of dNTPs.
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| 猪细环病毒2型环介导等温扩增检测方法的建立;张黎等;《中国畜牧兽医》;20150520;第42卷(第5期);第1098-1103页 * |
| 逆转录环介导等温扩增技术对新城疫病毒的快速检测;杜景娇;《中国优秀硕士学位论文全文数据库》;20140115(第1期);D050-304 * |
| 链球菌性乳房炎LAMP可视化检测方法的建立;贺婷等;《中国动物检疫》;20140430;第31卷(第4期);第85-89页 * |
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