CN106367529A - RT-LAMP kit for detecting American type highly-pathogenic porcine reproductive and respiratory syndrome by adopting rapid developing one-step method - Google Patents
RT-LAMP kit for detecting American type highly-pathogenic porcine reproductive and respiratory syndrome by adopting rapid developing one-step method Download PDFInfo
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Abstract
The invention provides an RT-LAMP kit for detecting American type highly-pathogenic porcine reproductive and respiratory syndrome by adopting a rapid developing one-step method. The kit comprises 6 RT-LAMP primers. For the kit, under the condition that the original reaction time, namely 1h, is not prolonged, the one-step operation of directly adding a fluorescent dye into a reaction tube is realized, therefore, the operation process is simplified, meanwhile, the fundamental problem of developing failure caused when the fluorescent dye is directly added into the reaction tube due to the inhibition of a premixing dye containing calcein for Bst DNA amplifier enzyme is solved, and moreover, the pollution that after the reaction is finished, a release product caused by secondary uncovering forms an aerosol is effectively avoided. With the adoption of the kit for carrying out detection, the direct and intuitive judgment through naked eyes can be realized, and in addition, the kit has good specificity and sensitivity.
Description
Technical Field
The invention relates to an RT-LAMP kit for detecting American highly pathogenic porcine reproductive and respiratory syndrome by a rapid chromogenic one-step method.
Background
Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as Porcine reproductive and respiratory syndrome, is a highly contagious swine epidemic disease that causes sow reproductive failure and dyspnea in piglets and finishing pigs due to an RNA virus, PRRSV. PRRSV can be divided into two genotypes, namely European (Eu) or type1 and North American (NA) or type2, based on antigenic and genomic characteristics. Since the 80's of the last century, PRRSV was discovered, this epidemic was widely prevalent in swine farms all over the world. The American type PRRSV has been popular in China for a long time. Unfortunately, in 2006, month 5, a highly pathogenic PRRSV identified as the American type (NA-HP-PRRS) began to emerge in Jiangxi province of China and rapidly spread throughout the country. Unlike the classical non-highly pathogenic viruses, the genome of the american type highly pathogenic PRRSV lacks almost continuously 90 bases in the non-structural protein-2, NSP2, coding region.
Since the emergence of American-type highly pathogenic PRRS in our country, the epidemic disease has been one of the most major epidemic diseases which plague the healthy development of the pig industry in China. Due to the lack of serological methods to distinguish infection from immunity, diagnosis of HP-PRRS relies primarily on nucleic acid detection methods, both conventional PCR and real-time quantitative PCR. Due to the limitation of instruments and high level requirements on operators, the methods are difficult to widely popularize and use in pig farms and primary epidemic prevention stations. At present, pig farm field diagnosis methods aiming at PPRS infection are still under development, and no corresponding commercial products are available.
The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method can realize isothermal expansion under the action of Bst DNA polymerase, and avoids the gradient lifting mode of conventional RT-PCR or real-time quantitative RT-PCR. Therefore, RT-LAMP can be completed in a common constant-temperature water bath and a heat block without expensive instruments and equipment. Furthermore, RT-LAMP can be judged by visual identification according to the change of the fluorescence color of the reaction tube. Therefore, RT-LAMP is expected to become a bedside rapid diagnosis method and is widely popularized and used on the basis.
The applicant establishes an RT-LAMP diagnostic method (Chen et al,2008) aiming at NA-HP-PRRS in 2008, the method designs 4 specific primers aiming at 6 target fragments in ORF1a area, amplification products can be detected by a typical trapezoidal strip formed by 2.5% agarose gel electrophoresis, and after the reaction is finished, a reaction tube cover is opened, SYBRGreen or a premixed fluorescent dye containing calcein and manganese ions is added, and the existence of NA-HP-PRRSV in a sample to be detected is judged according to the color change of the reaction tube. The positive reaction tubes developed a brown color upon addition of SYBR Green dye that could be visually identified, but the brown color was not as easily observable as the bright Green color formed by addition of the calci-ferous premixed dye. Probably because the calcium flavin has the effect of inhibiting Bst polymerase, the same primer pair is adopted, the premixed fluorescent dye containing the calcium flavin and the manganese ions is directly added into the reaction tube when the reaction solution is prepared, and under the conditions of the same temperature and amplification time, the positive sample tube can not generate the green fluorescence, so that the positive sample and the negative sample can not be distinguished. Even with agar gel electrophoresis, a classic trapezoidal band is difficult to observe. The two-step method of adding fluorescent dye for observation and judgment after the reaction is finished not only has complex operation, but also has the main problems that when the cover of the reaction tube is opened, an amplification product is released into the air to form a large amount of aerosol, the RT-LAMP method is very sensitive, the aerosol floating in the air can be completely used as a reaction template, so that false positive is formed in the subsequent operation, and the false positive rate is greatly increased. In order to simplify the operation steps, reduce the false positive rate to the maximum extent, fully ensure the advantages of observation and judgment by naked eyes, and meet the requirements of on-site detection of a pig farm and self-diagnosis of vast breeding manufacturers, further improvement is made. Around the characteristic NSP2 coding gene deletion region of the American type highly pathogenic PRRSV, 6 primers are designed, besides an inner primer pair and an outer primer pair, a loop primer pair is also introduced, the amplification efficiency is greatly improved, and the aim of one-step rapid display by directly adding a fluorescent dye into a reaction tube is fulfilled through repeated groping conditions. The field sample detection result shows that the newly established RT-LAMP detection method can quickly diagnose the American type highly pathogenic PRRS, has simple equipment, simple and convenient operation and good sensitivity and specificity, and is suitable for the field diagnosis of pig farms.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an RT-LAMP kit for detecting American type highly pathogenic porcine reproductive and respiratory syndrome by a rapid chromogenic one-step method. The field sample detection result shows that the RT-LAMP detection method can rapidly diagnose the American type highly pathogenic PRRS, has simple equipment, simple and convenient operation and good sensitivity and specificity, and is suitable for the field diagnosis of pig farms.
The invention provides a specific LAMP primer group for detecting American highly pathogenic porcine reproductive and respiratory syndrome, which comprises the following six primers:
F3：5'-GCTCCGCGCAGGAAGGTC-3'，
B3：5'-GCAAATCCAGAGGCTCATCC-3'，
FIP：5'-GGGCTCACTCATAGGTGTCATCGTAACGGTTCGGAAGAAACTGTC-3'，
BIP：5'-ACACCTTTGAGTGGGTCGGCACGTCAGCGTTGTTGTCACAGT-3'，
FLoop：5'-TTCCGACACCATCCGAGC-3'，
BLoop：5'-TCTACGCGGTGCAGGAAC-3'。
the invention also provides an RT-LAMP kit for detecting the American highly pathogenic porcine reproductive and respiratory syndrome by a rapid chromogenic one-step method, wherein the kit comprises the primer group in claim 1.
Preferably, the fluorescent dye also comprises a fluorescent dye containing calcein and manganese ions.
Preferably, the fluorescent dye is used in an amount of 0.4 to 0.5. mu.L in a 25. mu.L detection system.
Further preferably, the kit constitutes a LAMP detection system; the specific configuration of the 25 μ L detection system is:
PM premixed primer FIP/BIP (50UM) 0.4 muL
FLOOP/BLOOP (50UM) 0.4μL
F3/B3 (10UM) 0.2μL
1.0-2.0 μ L of extracted nucleic acid or RNA
Bst enzyme 1.5. mu.L
2×U-loadmix buffer 12.5μL
Rongyan Loopamp fluorescent dye containing calcein and manganese ions 0.4-0.5 μ L
DEPC-treated ddH2O Make up to 25.0. mu.L
Preferably, the detection reaction conditions of the kit are as follows: 60-65 ℃ for 45-90 minutes; the reaction was terminated.
Preferably, the detection reaction conditions of the kit are as follows: at 63-64 ℃ for 45-60 minutes; the reaction was terminated.
The invention also provides application of the primer group in preparation of a kit for detecting the American highly pathogenic porcine reproductive and respiratory syndrome.
According to the invention, 6 specific primers are designed aiming at 8 target fragments of the structural region according to the characteristic that the coding gene of the NA-HP-PRRSV non-structural protein NSP2 lacks 90 bases compared with the region of non-highly pathogenic PRRSV (Chen et al.2008), so that the amplification efficiency is greatly improved, a large number of amplification products are generated, and the inhibition rate of calcein is relatively reduced. Therefore, the premixed dye containing the calcium flavin is directly added into the reaction tube, and after the reaction is finished, the contrast color change can be directly observed, and the existence of NA-HP-PRRSV infection in the sample can be judged.
By introducing a pair of loop primers and searching for the optimal amplification temperature, the invention successfully establishes the NA-HP-PRRS rapid chromogenic one-step RT-LAMP detection method, and the detection results of a large amount of pig serum and tissue samples show that the method has strong sensitivity, nucleic acid extracted by cell toxicity is taken as a detection object, the minimum detection limit is 10 copies/reaction, and the sensitivity is 10000 to 100000 times higher than that of the conventional RT-PCR method. The newly established RT-LAMP adopts 3 pairs of specific primers aiming at the NA-HP-PRRSV gene identification region, so that the RT-LAMP has strong specificity and does not have cross reaction with Non-HP-PRRSV, EU-PRRSV, PCV1, PCV2, PRV and CSF. Further, by detecting a large number of clinical samples and comparing with a conventional RT-PCR detection method, the NA-HP-PRRSV rapid chromogenic one-step RT-LAMP is not only strong in specificity, but also high in clinical sensitivity. The serum, the lung and the lymph node are all suitable for rapid chromogenic one-step RT-LAMP detection, and particularly, the serum sample is convenient to collect and beneficial to general investigation. Therefore, the NA-HP-PRRSV rapid color development one-step method RT-LAMP has wide market application prospect, can be developed into a large-scale general inspection method, and is expected to be developed into a rapid diagnosis kit which can be used in a large number of pig farm sites and basic epidemic prevention stations.
The advantages of simple equipment requirements (a constant-temperature water bath kettle and a hot block), convenient one-step operation process, direct visual judgment by naked eyes, high sensitivity (10 copy number RNA or plasmid/reaction can be detected), and strong specificity endow the American type highly pathogenic blue ear disease with independent charm of the RT-LAMP in a rapid color development one-step method, and the method is suitable for being used for detecting and generally surveying the infection conditions of the NA-HP-PRRSV virus of the swinery and wide basic epidemic prevention stations, and has wide market application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a diagram of RT-PCR (P224/P225 primer) amplified PRRSV agarose gel electrophoresis;
FIG. 2 is a NA-HP-PRRSV rapid color development one-step RT-LAMP map under different temperature conditions of 60-65 ℃;
FIG. 3 shows the result of performing a one-step RT-LAMP detection on NA-HP-PRRSV by taking RNA of a 10-fold serially diluted NA-HP-PRRSV QH08 strain as a template;
FIG. 4 shows the detection of the analytical sensitivity of RT-PCR (P224/P225 primer) using 10-fold serial dilution of NA-HP-PRRSV QH08 strain RNA as a template (4A) and using the purified DNA fragment as a template (4B);
FIG. 5 shows the result of the analysis specificity detection of the NA-HP-PRRSV rapid color development one-step RT-LAMP.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
1. Strain
The NA-HP-PRRSV (American type highly pathogenic porcine reproductive and respiratory syndrome virus) QH08 strain was isolated and stored by the laboratory, and genome-wide sequencing Genebank accession number KU 201579. The NA-Non-HP-PRRSV (American type Non-highly pathogenic porcine reproductive and respiratory syndrome Virus) reference strains are CH-1R and CH-1 a. Porcine parvovirus type1 (PPV 1) AV30 strain and AV31 strain, porcine circovirus type1 (PCV 1) CAU0672 strain, porcine circovirus type2 (PCV 2) CAU0673 strain, porcine pseudorabies virus (RRV) AV250 strain and classical swine fever virus (CSF) AV65 strain.
The european type PRRSV SE strain was from the european triangle research centre of hungarian and the european type PRRSV PL strain was from the porcine reproductive and respiratory syndrome OIE reference laboratory provided RNA samples from the national veterinary institute of polish.
2. Clinical samples
The field samples for clinical detection are from pig farms in different areas of Gansu province, and comprise 596 samples, wherein the sample comprises 129 pig sera and 467 tissue samples. 129 pig sera were positive in 52 and negative in 77. The tissue samples comprise 82 parts of lung tissues (43 parts of positive and 39 parts of negative), 68 parts of liver tissues (36 parts of positive and 32 parts of negative), 68 parts of heart tissues (33 parts of positive and 35 parts of negative), 88 parts of kidney tissues (47 parts of positive and 41 parts of negative), 71 parts of spleen tissues (35 parts of positive and 36 parts of negative) and 90 parts of lymph nodes (45 parts of positive and 45 parts of negative), and after nucleic acids of the samples are extracted, the NA-HP-PRRSV rapid chromogenic one-step RT-LAMP and conventional RT-PCR detection are respectively carried out for detecting the clinical specificity and sensitivity of a newly established method.
3. Nucleic acid amplification and extraction related kit
The amplification kit for establishing the RT-LAMP method is an RT-LAMP kit of Beijing Meilebo medical science and technology Limited company and a Loopamp fluorescence visual detection kit (containing calcium flavin and manganese ions) of Japan Rongyan company. The nucleic acid was extracted using TaKaRa MiniBEST Viral RNA/DNA extraction kit Ver.5.0, manufactured by Takara Bio-engineering (Dalian) Ltd. The kit for conventional PCR amplification was derived from PremixTaq Version 2.0 plus dye, manufactured by Takara Bio engineering (Dalian) Ltd. The Kit used for RT-PCR was PrimeScriptTM One StepRT-PCR Kit Ver.2 from Takara.
Example 1
Firstly, extracting nucleic acid of sample
The nucleic acids in the cytotoxic, serum and ground tissue samples were extracted using the MiniBEST Viral RNA/DNA extraction kit from Takara Bio-engineering (Dalian) Ltd.
Second, conventional RT-PCR reaction experiment determination of NA-PRRSV
1. Primers for RT-PCR reaction
TABLE 1 NA-PRRSV conventional RT-PCR amplification primer sequences
P224/P225 is an outer primer pair (Chen et al.2008) of a classical RT-LAMP method established in 2008 by the applicant, the size of an amplified fragment is predicted to be 246bp for NA-HP-PRRSV, and the size is predicted to be 336bp for NA-Non-HP-PRRSV.
2. RT-PCR amplification reaction system
RT-PCR amplification was performed using the PrimeScriptTM One Step RT-PCR Kit Ver.2, Takara.
RT-PCR reaction system:
PrimeScript 1 Step Enzyme Mix 0.8μl
2×1 Step buffer 10μl
Primer-F（20μM） 1μl
Primer-R（20μM） 1μl
template 1. mu.l
DEPC water was added to 20. mu.l.
3. RT-PCR amplification reaction process
And (3) amplification procedure: at 50 ℃ for 30 min; 94 ℃ for 5 min; (94 ℃, 30 s; 58 ℃, 30 s; 72 ℃, 40s/90 s; 36 cycles); 72 ℃ for 10 min.
4. Electrophoresis detection of RT-PCR amplification product
RT-PCR amplification products were detected by conventional agarose gel electrophoresis, with a gel concentration of 1%, 1XTAE in electrophoresis buffer, and an electrophoresis speed of 5-6 volts/cm (i.e., voltage drop).
Third, establishing of NA-HP-PRRSV fast color development one-step method RT-LAMP
1. NA-HP-PRRSV rapid chromogenic one-step RT-LAMP amplification primer selection and sequence
The applicant analyzes and compares the whole genome sequences of American type highly pathogenic PRRSV strains, American type non-highly pathogenic PRRSV strains and European type PRRSV strains, and adopts DNAstar software and alignment to design specific primers around 90 base deletion regions of NSP2 non-structural coding genes of NA-HP-PRRSV, aiming at distinguishing the three types of PRRSV.
The NA-HP-PRRSV fast chromogenic one-step RT-LAMP has 6 primers in total (see Table 2), and in addition to a conventional pair of outer primers (F3 and B3) and a pair of inner primers (FIP and BIP), there is a pair of loop primers, i.e., FLooP and BLOOP. The purpose of introducing the loop primer is to further improve the amplification efficiency and the yield and facilitate quick color development.
TABLE 2 HP-PRRSV Rapid chromogenic one-step LAMP primer sequence
2. RT-LAMP amplification reaction system
The reaction system of NA-HP-PRRSV RT-LAMP is 25 mu L/tube, and the sample loading is as follows:
PM premixed primer FIP/BIP (50UM) 0.4 muL
FLOOP/BLOOP (50UM) 0.4 μL
F3/B3 (10UM 0.2 μL
And (3) RNA extraction: 1.0-2.0 mu L
Bst enzyme 1.5. mu.L
2×U-loadmix buffer： 12.5 μL
Rongyan Loopamp fluorescent dye (containing calcein and manganese ions) 0.4-0.5 μ L
DEPC-treated ddH2O to 25.0. mu.L
The total volume was 25.0. mu.L.
The RT-LAMP reaction relates to 6 primers, and the 6 primers are premixed according to the amount before the experiment to prepare premixed primers, and the operation is carried out according to the calculation of (1 mu L premixed primers)/tube. The amount of the fluorescent dye containing calcein may be added to the reaction system/tube in an amount of 0.4-0.5. mu.L/25. mu.L, which reduces the cost on the one hand and the inhibition of the amplification reaction by the fluorescent dye containing calcein on the other hand.
3. RT-LAMP amplification reaction process
RT-LAMP is isothermal amplification reaction, so PCR amplification instrument is not needed, only instrument capable of providing isothermal reaction is needed, and the most common instrument is common water bath or hot block. And (3) instantly centrifuging the reaction tube after sample application, and then putting the reaction tube into a water bath (the tube can be inserted into a buoy) or a hot block.
And (3) amplification procedure: reacting for 45-90 minutes at 60-65 ℃. Typically 60 minutes. The reaction effect is best at 63 ℃ or 64 ℃.
4. Electrophoresis detection of RT-LAMP amplification product
(1) Agarose gel electrophoresis detection method
RT-LAMP amplification products can be detected by adopting the conventional agarose gel electrophoresis, the gel concentration is 2-2.5%, the electrophoresis buffer solution is 1XTAE, and the electrophoresis speed is 5-6 volts/cm (namely voltage drop). After electrophoresis, the gel was stained in a stain containing EB for 30 minutes, and then observed under an ultraviolet lamp and photographed by a computer.
(2) Fluorescent visual inspection method
The rapid chromogenic one-step RT-LAMP has the advantages that since the dye containing the calcium flavin and the manganese ions is added into the reaction tube, bright green fluorescence appears in the positive sample tube after the reaction is finished, and only weak background fluorescence exists in the control tube and the negative sample tube. Thus, by comparing with the negative and positive control set by the system, under the natural light, the judgment can be made by visual observation. Furthermore, the positive samples produced a larger amount of product due to amplification, so the positive tube solution was more turbid than the negative control. In a laboratory, the color change can be observed under an ultraviolet lamp, and the color change is more obvious than that observed under natural light.
Fourth, Performance testing of the method of the invention
1. NA-HP-PRRSV rapid color development one-step RT-LAMP analysis sensitivity experiment
RNA is extracted from Mark-145 cytotoxin of NA-NA-HP-PRRSV QH08 strain, the concentration of nucleic acid is measured by an ultraviolet spectrophotometer, and then the copy number of nucleic acid per microliter of an extracted RNA sample is calculated according to the molecular weight of nucleic acid of a whole genome (Genebank accession number KT 033733). RNA samples were treated with sterile and nuclease-free ddH2O is serially diluted to 1 × 10^ 6-1 × 10^0 (nucleic acid copy number)/mu L, then RT-LAMP experiments are carried out according to the method, 1 mu L of the serially diluted RNA sample is added into each reaction tube to be used as a template, agarose gel electrophoresis and fluorescence detection are carried out after the reaction is finished, the RNA samples diluted by times are simultaneously subjected to conventional RT-PCR amplification, and the analysis sensitivity of the two methods is compared.
2. NA-HP-PRRSV rapid color development one-step RT-LAMP analysis specificity experiment
In testing the analytical specificity of the method, cross-reactivity experiments are respectively carried out with other common viruses such as NA-Non-PRRSV, EU-PRRSV, PCV1, PCV2, PPV1, PRV and CSF which can cause the occurrence of the porcine reproductive disorder diseases. The specific strains are PCV1 (CAU 0672), PCV2 (CAU 0673), PPV1 type AV30 strain and AV31 strain, PRV AV250 strain, CSF AV65 strain and NA-Non-HP-PRRSV CH-1R strain. The European PRRSV SE strain is from the European triangle research center of Hungarian, and the European PRRSV PL strain is from the OIE reference laboratory of porcine reproductive and respiratory syndrome of the national veterinary institute of Poland, both of which are RNA samples.
3. NA-HP-PRRSV rapid color development one-step RT-LAMP clinical sensitivity experiment
The positive sample for NA-HP-PRRSV rapid display one-step RT-LAMP clinical sensitivity analysis is from tissue and serum samples of pig farms in different areas of Gansu province with reproductive disturbance diseases. 52 parts of serum, 43 parts of lung tissue, 36 parts of liver tissue, 33 parts of heart tissue, 47 parts of kidney tissue and 35 parts of spleen tissue; lymph node 45 parts, total 291. These samples were determined to be NA-HP-PRRSV infected by a comprehensive assay such as ELISA, cell-mediated detoxification, etc. After RNA of the samples is extracted, the NA-HP-PRRSV rapid display one-step RT-LAMP and conventional RT-PCR detection are respectively carried out.
4. NA-HP-PRRSV rapid color development one-step RT-LAMP clinical specificity experiment
The negative samples for the NA-HP-PRRSV rapid chromogenic one-step RT-LAMP clinical specificity analysis are from healthy pig farms in different areas of Gansu province, and comprise 305 samples, wherein 77 parts of serum and 228 parts of tissue samples. 39 parts of lung tissue, 32 parts of liver tissue, 35 parts of heart tissue, 41 parts of kidney tissue and 36 parts of spleen tissue in the tissue sample; 45 parts of lymph node. After nucleic acids of the samples are extracted, NA-HP-PRRSV rapid chromogenic one-step RT-LAMP and conventional RT-PCR detection are respectively carried out, and the clinical specificity of the newly established rapid chromogenic one-step RT-LAMP is evaluated.
[ results ] A method for producing a compound
1. Conventional RT-PCR (reverse transcription-polymerase chain reaction) method for detecting NA-HP-PRRSV (porcine reproductive and respiratory syndrome Virus)
A P224/P225 primer pair designed around a 90-base deletion region of an NA-HP-PRRSV Non-structural protein NSP2 encoding gene is adopted to amplify RNA templates of an NA-HP-PRRSV strain QH08, a Non-HA-PRRSV strain CH-1R and an EU-PRRSV strain SE respectively, and the result is shown in figure 1. Electrophoresis pattern shows that P224/P225 primer amplification is adopted, cross reaction with EU-PRRSV is avoided, the size of the NA-HP-PRRSV amplified fragment is predicted to be 246bp, and the size of the NA-Non-HP-PRRSV amplified fragment is predicted to be 336 bp.
FIG. 1 is a diagram of RT-PCR (P224/P225 primer) amplified PRRSV agarose gel electrophoresis; wherein, M is DL2000molecular marker (2000,1000,750,500,250,100 bp); RNA of EU-PRRSV strain SE; 2. NA-NA-HP-PRRSV QH08 cytotoxic RNA; 3. NA-Non-HP-PRRSV CH-1R cytotoxic RNA.
2. NA-HP-PRRSV rapid color development one-step RT-LAMP establishment
The result of establishing the NA-HP-PRRSV rapid color development one-step RT-LAMP by taking the cytotoxic RNA of the NA-NA-HP-PRRSV QH08 strain as a template shows that a typical trapezoidal strip can be effectively amplified at 60-65 ℃, and the green fluorescence of a reaction tube is bright under the ultraviolet mode of a gel imager ultraviolet LAMP. The bands are more clearly evident at 63 and 64 ℃, and the fluorescence color of the reaction tube is very strong and brighter. In the case of natural light, the green fluorescence is visible to the naked eye as a function of temperature. The photographing effect is not as good as the effect observed by naked eyes, as shown in fig. 2. As can be seen from the results in FIG. 2, the suitable amplification temperatures for the HP-PRRSV rapid chromogenic one-step RT-LAMP were 63 ℃ and 64 ℃.
FIG. 2 is a HP-PRRSV rapid color development one-step RT-LAMP map under different temperature conditions of 60-65 ℃; wherein,
a, picture A: agarose gel electrophoresis pattern of LAMP amplification product;
and B, drawing: photographing a map of the LAMP amplification product corresponding to the reaction tube in an ultraviolet mode of the gel imager ultraviolet light;
and (C) diagram: photographing the map of the LAMP amplification product corresponding to the reaction tube in a black and white mode of the gel imager ultraviolet light;
and (D) diagram: photographing a map of the LAMP amplification product corresponding to the reaction tube under the condition of natural light;
m: DL2000 Marker (2000,1000,750,500,250,100 bp); 1,2,3,4,5,6 represent isothermal amplifications at 60, 61, 62, 63, 64, 65 ℃ respectively.
3. NA-HP-PRRSV rapid chromogenic one-step RT-LAMP analytical sensitivity detection
The whole gene of the NA-NA-HP-PRRSV QH08 is 15349bp in length, has the average molecular weight of 340 daltons according to the ssRNA each base, has the average molecular weight MW of 5218660 daltons, and contains the gene copy number of 1.15x10^8/μ l when the RNA concentration is 1ng/μ l. After 10 times of serial dilution, rapid chromogenic one-step LAMP (isothermal amplification at 65 ℃) and conventional RT-PCR detection are carried out, and the sensitivities of the two types are compared.
As a result, when the RNA nucleic acid extracted by the cell virus was used as a detection target, the LAMP detection limit of the fast chromogenic one-step method was 10 copies/. mu.L (FIG. 3), and the detection limit of the conventional RT-PCR method (P224/P225 primer pair) was about 1x10^6 copies/. mu.L (FIG. 4A). Using the purified product of P224/P225 amplification (QH 08 RNA as template) as template, and performing 10-fold serial dilution at 1-10^8, the detection limit of the conventional RT-PCR method is 100 copies/. mu.L (FIG. 4B), while the LAMP method is still 10 copies/. mu.L (refer to FIG. 3, because the pattern band is unchanged). This indicates that one-step rapid chromogenic RT-LAMP is more sensitive than conventional RT-PCR, and can be increased by about 10 times (using purified DNA fragment as template) and 100000 times (using RNA as template).
FIG. 3 shows the result of HP-PRRSV rapid color development one-step RT-LAMP detection using 10-fold serial dilution NA-HP-PRRSV QH08 strain RNA as a template; wherein,
a, picture A: agarose gel electrophoresis pattern of RT-LAMP amplification product;
and B, drawing: photographing a map of the RT-LAMP amplification product corresponding to the reaction tube in an ultraviolet mode of the gel imager ultraviolet light;
and (C) diagram: photographing the map of the RT-LAMP amplification product corresponding to the reaction tube in a black and white mode of the gel imager ultraviolet light;
m: DL2000 Marker (2000,1000,750,500,250,100 bp); the corresponding template copy numbers (copy number/. mu.L) are 1(0) respectively; 2 (1 x10^ 0); 3 (1 x10^ 1); 4(1 x10^ 2); 5(1 x10^ 3); 6(1 x10^ 4); 7(1 x10^ 5); 8(1 x10^ 6); 9(1 x10^ 7); 10(1 x10^ 8).
FIG. 4 shows the detection of the analytical sensitivity of RT-PCR (P224/P225 primer) using 10-fold serial dilution of NA-HP-PRRSV QH08 strain RNA as a template (4A) and using the purified DNA fragment as a template (4B); wherein,
panel A is the template RNA and panel B is the template purified DNA fragment.
M: DL2000 Marker; lanes 1-10 RNA template copy number 0-1x10^ 8.
4. Analysis specificity of NA-HP-PRRSV rapid color development one-step RT-LAMP
The newly established NA-HP-PRRSV rapid chromogenic one-step RT-LAMP has strong specificity, only reacts with NA-HP-PRRSV reference strains (TJM-F92, HUN4-F112, JXA1-R, HPBEDV and LNXM), and does not cross-react with NA-Non-HP-PRRSV reference strains (CH-1R and CH-1 a), EU-PRRSV reference strains (SE and PL) and PCV1 (CAU 0672), PCV2 (CAU 0673), AV30 strain of PPV1 type (CVCCAV 30), PRV (CVCC AV 250) and CSF (CVCCAV 65) (see figure 5).
FIG. 5 shows the result of the analysis specificity detection of the NA-HP-PRRSV rapid color development one-step RT-LAMP; wherein,
a, an agarose gel electrophoresis pattern of an RT-LAMP isothermal amplification product;
b, taking a picture of the RT-LAMP isothermal amplification product corresponding to the reaction tube in a black and white mode of the gel imager ultraviolet light;
c, taking a picture of the reaction tube corresponding to the RT-LAMP isothermal amplification product in an ultraviolet mode of the ultraviolet light of a gel imager;
m: DL2000 Marker (2000,1000,750,500,250,100 bp); nucleic acids of different viruses are taken as templates, and the method sequentially comprises the following steps: (1) ddH2O; (2) NA-NA-HP-PRRSV QH08 strain; (3) NA-HP-PRRSV TJM-F92 strain; (4) NA-HP-PRSSVHUN4-F112 strain; (5) NA-HP-PRRSV JXA1-R strain; (6) the NA-HP-PRRSV HPBEDV strain; (7) NA-HP-prrsv vlnxm strain; (8) Non-HP-PRRSV CH-1R strain; (9) Non-HP-PRRSV CH-1a strain; (10) the AV30 strain PPV1 type (CVCCAV 30); (11) PCV1 (CAU 0672); (12) PCV2 (CAU 0673); (13) PRV (CVCC AV 250); (14) the kit provides a positive control; (15) CSF (CVCCAV 65); (16) EU-PRRSV SE strain; (17) EU-PRRSV PL strain.
5. Clinical sensitivity detection of NA-HP-PRRSV rapid chromogenic one-step RT-LAMP
291 parts of HP-PRRSV positive clinical samples are adopted to detect the clinical sensitivity of NA-HP-PRRSV rapid color development one-step RT-LAMP, and the comparison analysis is carried out with the conventional RT-PCR. The results in Table 3 show that the total positive detection rate of the RT-LAMP method is 96.2%. The total detection rate of the conventional RT-PCR amplified by the P224/P225 primer pair is 90.4%. For pig serum, the detection rate of the RT-LAMP method is 96.2%, and the detection rate of the RT-PCR method is 90.4%. For tissue samples, the detection rate of RT-LAMP is 90.9-100%, and the detection rate of RT-PCR is 84.8-95.3%. The results show that the newly established NA-HP-PRRSV quickly shows that the clinical sensitivity of the one-step RT-LAMP is obviously higher than that of the RT-PCR, has high detection rate on a serum sample, and is suitable for clinical living body general detection.
TABLE 3291 Rapid color development one-step RT-LAMP and RT-PCR detection sensitivity comparison of NA-HP-PRRSV positive clinical samples
6. Clinical specificity detection of NA-HP-PRRSV rapid chromogenic one-step RT-LAMP
Clinical specificity analysis of RT-LAMP was performed on samples collected from pig farms where no porcine reproductive obstructive disease had occurred, and compared with RT-PCR. A total of 305 samples were tested, 77 serum samples and 228 tissue samples. For these negative clinical samples, the total detection rate of one-step RT-LAMP was 95.4% and the total detection rate of conventional RT-PCR method was 93.1%. The detection rates for the serum samples were 97.4% (RT-LAMP) and 93.1% (RT-PCR), respectively. The detection rate of RT-LAMP on tissue samples is 93.4% -97.4%, and the detection rate of RT-PCR is (90.6-94.8%). Both methods have good specificity, but the specificity of the rapid chromogenic one-step RT-LAMP is higher than that of the RT-PCR method. The lung, lymph nodes and blood are all suitable for being used as samples for detection by RT-LAMP and RT-PCR methods. The newly established NA-HP-PRRSV rapid chromogenic one-step method has strong LAMP specificity, can detect various tissue samples including serum, and is suitable for clinical popularization and use.
TABLE 4267 quick color development of NA-HP-PRRSV in negative clinical samples comparing specificity of RT-LAMP and RT-PCR method in one-step method
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The specific RT-LAMP primer group for detecting the American highly pathogenic porcine reproductive and respiratory syndrome is characterized by comprising the following components in parts by weight: the primer group comprises the following six primers:
F3：5'-GCTCCGCGCAGGAAGGTC-3'，
B3：5'-GCAAATCCAGAGGCTCATCC-3'，
FIP：5'-GGGCTCACTCATAGGTGTCATCGTAACGGTTCGGAAGAAACTGTC-3'，
BIP：5'-ACACCTTTGAGTGGGTCGGCACGTCAGCGTTGTTGTCACAGT-3'，
FLoop：5'-TTCCGACACCATCCGAGC-3'，
BLoop：5'-TCTACGCGGTGCAGGAAC-3'。
2. an RT-LAMP kit for detecting American highly pathogenic porcine reproductive and respiratory syndrome by a rapid chromogenic one-step method is characterized in that: the kit comprises the primer set of claim 1.
3. The kit of claim 2, wherein: also included are fluorescent dyes containing calcein and manganese ions.
4. The kit of claim 3, wherein: the dosage of the fluorescent dye is 0.4-0.5 mu L in a 25 mu L detection system.
5. The kit according to any one of claims 2 to 4, wherein: the kit forms an RT-LAMP detection system; the specific configuration of the 25 μ L detection system is:
PM premixed primer FIP/BIP (50UM) 0.4 muL
FLOOP/BLOOP (50UM) 0.4μL
F3/B3 (10UM) 0.2μL
1.0-2.0 μ L of extracted nucleic acid or RNA
Bst enzyme 1.5. mu.L
2×U-loadmix buffer 12.5μL
Rongyan Loopamp fluorescent dye containing calcein and manganese ions 0.4-0.5 μ L
DEPC-treated ddH2O was supplemented to 25.0. mu.L.
6. The kit of claim 5, wherein: the detection reaction conditions of the kit are as follows: 60-65 ℃ for 45-90 minutes; the reaction was terminated.
7. The kit of claim 6, wherein: the detection reaction conditions of the kit are as follows: at 63-64 ℃ for 45-60 minutes; the reaction was terminated.
8. Use of the primer set of claim 1 in the preparation of a kit for detecting the American-type highly pathogenic porcine reproductive and respiratory syndrome.
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| CN105861754A (en) * | 2016-05-30 | 2016-08-17 | 中国农业科学院兰州兽医研究所 | Multiple RT-PCR detection kit for American type porcine reproductive and respiratory syndrome virus and porcine parvovirus |
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| CN101575650A (en) * | 2009-06-24 | 2009-11-11 | 中国兽医药品监察所 | Porcine reproductive and respiratory syndrome virus RT-LAMP detection kit and detection method thereof |
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