CN106367529B - A rapid chromogenic one-step RT-LAMP kit for the detection of American type highly pathogenic porcine reproductive and respiratory syndrome - Google Patents

Abstract

The present invention provides the RT-LAMP kit of a kind of detection of quick colour-developing one-step method american type high-pathogenicity porcine reproductive and respiration syndrome, which includes 6 RT-LAMP primers.Kit of the invention is not under conditions of extending original 1 small the reaction time, realize the single stepping that fluorescent dye is directly added into reaction tube, not only simplify operation sequence, solve the premix dyestuff containing calcium flavin causes to be directly added into the root problem that fluorescent dye does not develop the color in reaction tube to the inhibition of Bst DNA cloning enzyme, also effectively prevents since the secondary pollution that caused release product forms aerosol of uncapping occurs after reaction.Detected using kit of the invention, can the direct intuitive judgment of naked eyes, there is good specificity and sensibility.

Description

A rapid chromogenic one-step assay for the detection of reproductive and respiratory syndrome in highly pathogenic pigs of American type Sign's RT-LAMP kit

technical field

The invention relates to a RT-LAMP kit for the rapid color development one-step detection of American type highly pathogenic porcine reproductive and respiratory syndrome.

Background technique

Porcine reproductive and respiratory syndrome (PRRS), commonly known as porcine blue-ear disease, is a highly contagious swine disease caused by an RNA virus, namely PRRSV, which causes reproductive disorders in sows and breathing difficulties in piglets and fattening pigs. According to antigenicity and genomic characteristics, PRRSV can be divided into two genotypes, namely European type (European, Eu) or type1 and North American (North American, NA) or type2. Since the discovery of PRRSV in the 1980s, the disease has spread widely on pig farms around the world. The American type PRRSV has been popular in my country for a long time. Unfortunately, in May 2006, a type of highly pathogenic PRRSV (NA-HP-PRRS) identified as the American type began to appear in Jiangxi Province, my country, and spread rapidly throughout the country. Different from the classical non-highly pathogenic viruses in the past, the genome of the American type highly pathogenic PRRSV has almost a continuous deletion of 90 bases in the coding region of the non-structural protein-2, namely NSP2.

Since the outbreak of the American type highly pathogenic PRRS in my country, the disease has been one of the most important diseases that plagued the healthy development of my country's pig industry. Due to the lack of serological methods to distinguish infection from immunity, the diagnosis of HP-PRRS mainly relies on nucleic acid detection methods such as conventional PCR and real-time quantitative PCR. Due to the limitations of instruments and high-level requirements for operators, these methods are difficult to be widely used in pig farms and grassroots epidemic prevention stations. At present, on-site diagnostic methods for PPRS infection in pig farms are still under development, and no corresponding commercial products have come out.

The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method can achieve isothermal expansion under the action of Bst DNA polymerase, avoiding the need for conventional RT-PCR or real-time quantitative RT-PCR. Gradient lift mode. Therefore, RT-LAMP does not require expensive equipment and can be completed in an ordinary constant temperature water bath and heat block. In addition, RT-LAMP can make judgments by visual recognition according to the change of fluorescent color of the reaction tube. Therefore, RT-LAMP is expected to become a rapid diagnosis method at the bedside, and it will be widely used in the foundation.

The applicant established the RT-LAMP diagnostic method for NA-HP-PRRS in 2008 (Chen et al, 2008). This method designs 4 specific primers for the 6 target fragments in the ORF1a region, and the amplified product can be The typical ladder-shaped bands formed by 2.5% agarose gel electrophoresis can be detected. After the reaction is completed, open the lid of the reaction tube and add SYBRGreen or a premixed fluorescent dye containing calcein and manganese ions. According to the color change of the reaction tube, judge the waiting time Check the sample for the presence of NA-HP-PRRSV. Positive reaction tubes can develop a brown color after the addition of SYBR Green dye, which can be visually recognized, but the brown color is not as easy to observe as the bright green color formed by the addition of calcein-containing premixed dyes. It may be because calcein has the effect of inhibiting Bst polymerase. Using the same primer pair, the pre-mixed fluorescent dye containing calcein and manganese ions was directly added to the reaction tube when preparing the reaction solution. Under the same temperature and amplification time conditions , the positive sample tube cannot produce the above-mentioned green fluorescence, and cannot distinguish between positive and negative samples. Even with agarose gel electrophoresis, the classic ladder-shaped band is difficult to observe. The two-step method of adding fluorescent dyes for observation and judgment after the reaction is completed is not only cumbersome to operate, but also the main problem is that when the lid of the reaction tube is opened, the amplification product is released into the air, forming a large amount of aerosol, RT- The LAMP method is very sensitive, and the aerosol floating in the air can be used as a reaction template, which leads to the formation of false positives in subsequent operations, and the false positive rate is greatly increased. In order to simplify the operation steps, reduce the false positive rate as much as possible, fully guarantee the advantages of naked eye observation and judgment, and meet the needs of on-site inspection of pig farms and the need for self-diagnosis of the majority of breeders, we have made further improvements. Six primers were designed around the characteristic deletion region of the NSP2-encoding gene of the American type highly pathogenic PRRSV. In addition to the inner and outer primer pairs, we also introduced a loop primer pair, which greatly improved the amplification efficiency. The fluorescent dye is directly added to the reaction tube for the purpose of one-step rapid display. The field sample detection results show that the newly established RT-LAMP detection method can quickly diagnose the American type highly pathogenic PRRS, with simple equipment, simple operation, good sensitivity and specificity, and is suitable for on-site diagnosis of pig farms.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the prior art, the present invention provides an RT-LAMP kit for rapid color development one-step detection of American type highly pathogenic porcine reproductive and respiratory syndrome. The field sample detection results show that the RT-LAMP detection method of the present invention can rapidly diagnose the American type highly pathogenic PRRS, has simple equipment, simple operation, good sensitivity and specificity, and is suitable for on-site diagnosis of pig farms.

The present invention provides a specific LAMP primer set for detecting American type highly pathogenic porcine reproductive and respiratory syndrome, and the primer set is 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 present invention also provides a RT-LAMP kit for rapid color development one-step detection of American type highly pathogenic porcine reproductive and respiratory syndrome, the kit comprising the primer set of claim 1 .

Preferably, fluorescent dyes containing calcein and manganese ions are also included.

Preferably, the amount of the fluorescent dye is 0.4-0.5 μL in a 25 μL detection system.

As a further preference, the kit constitutes a LAMP detection system; the specific configuration of the 25 μL detection system is:

PM Premix Primer: FIP/BIP (50UM) 0.4μL

FLOOP/BLOOP (50UM) 0.4μL

F3/B3 (10UM) 0.2μL

Extracted nucleic acid or RNA 1.0-2.0μL

Bst enzyme 1.5μL

2×U-load mix buffer 12.5μL

Eiken Loopamp Fluorescent Dye with Calcein and Manganese 0.4-0.5μL

DEPC-treated ddH ₂ O supplemented to 25.0 μL

Preferably, the detection reaction conditions of the kit are: 60-65° C., 45-90 minutes; termination of the reaction.

Preferably, the detection reaction conditions of the kit are: 63-64° C., 45-60 minutes; termination of the reaction.

The present invention also provides the application of the above-mentioned primer set in preparing a kit for detecting the American type highly pathogenic porcine reproductive and respiratory syndrome.

According to the feature that the coding gene of NA-HP-PRRSV non-structural protein NSP2 is missing 90 bases compared with the non-high pathogenic PRRSV region (Chen et al. 2008), the present invention is designed for 8 target fragments of this structural region Six specific primers were used, which greatly improved the amplification efficiency, produced a large number of amplification products, and relatively reduced the inhibition rate of calcein. Therefore, the premixed dye containing calcein is directly added to the reaction tube, and after the reaction is completed, the color change of the contrast can be directly observed to determine the presence or absence of NA-HP-PRRSV infection in the sample.

By introducing a pair of loop primers and exploring the optimal amplification temperature, the present invention has successfully established the NA-HP-PRRS rapid color development one-step RT-LAMP detection method. The detection results of a large number of pig serum and tissue samples show that the method of the present invention has the advantages of It has strong sensitivity. Taking the nucleic acid extracted by cytotoxicity as the detection object, its minimum detection limit is 10 copies/reaction, and its sensitivity is 10,000 or even 100,000 times higher than that of conventional RT-PCR methods. The newly established RT-LAMP uses 3 pairs of specific primers for the NA-HP-PRRSV gene identification region, so it has strong specificity and does not interact with Non-HP-PRRSV, EU-PRRSV, PCV1, PCV2, PRV and CSF. Cross-reaction occurs. Furthermore, the detection of a large number of clinical samples and the comparison with conventional RT-PCR detection methods showed that NA-HP-PRRSV rapid color development one-step RT-LAMP not only has high specificity, but also has high clinical sensitivity. Serum, lung, and lymph nodes are all suitable for rapid chromogenic one-step RT-LAMP detection, especially for serum samples, which are convenient to collect and facilitate census investigation. Therefore, the NA-HP-PRRSV rapid color development one-step method RT-LAMP of the present invention has a broad market application prospect, can be developed as a large-scale general inspection method, and is expected to be developed as a rapid coloring method that can be used in the majority of pig farms and grassroots epidemic prevention stations. Diagnostic kits.

Simple equipment requirements (thermostatic water bath and heat block), convenient one-step operation process, direct and intuitive judgment of the naked eye, high sensitivity (can detect 10 copies of RNA or plasmid/reaction) and strong specificity give the advantages American type highly pathogenic porcine PRRSV rapid color development one-step RT-LAMP independent charm, this method is suitable for pig farms and the majority of grass-roots epidemic prevention stations to detect and census the infection status of NA-HP-PRRSV virus in pig herds. Broad market application prospects.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification, and are used to explain the present invention together with the embodiments of the present invention, and do not constitute a limitation to the present invention. In the attached image:

Figure 1 shows the agarose gel electrophoresis pattern of PRRSV amplified by RT-PCR (P224/P225 primers);

Figure 2 is the RT-LAMP spectrum of NA-HP-PRRSV rapid color development one-step method under different temperature conditions of 60-65 °C;

Figure 3 shows the results of 10-fold serial dilution of NA-HP-PRRSV QH08 strain RNA as a template, and the results of NA-HP-PRRSV rapid color development one-step RT-LAMP detection;

Figure 4 shows the analytical sensitivity detection of RT-PCR (P224/P225 primers) with 10-fold serial dilution of NA-HP-PRRSV QH08 strain RNA as template (4A) and purified DNA fragment as template (4B);

Figure 5 shows the analytical specificity detection results of NA-HP-PRRSV rapid color development one-step RT-LAMP.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.

1. Strain

NA-HP-PRRSV (American type highly pathogenic porcine reproductive and respiratory syndrome virus) QH08 strain was isolated and preserved by the laboratory, and the whole genome was sequenced with Genebank accession number KU201579. NA-Non-HP-PRRSV (American non-highly pathogenic porcine reproductive and respiratory syndrome virus) reference strains are CH-1R and CH-1a. Porcine parvovirus type 1 (PPV1) AV30 and AV31 strains, porcine circovirus type 1 (PCV1) CAU0672 strain, porcine circovirus type 2 (PCV2) CAU0673 strain, porcine pseudorabies virus (RRV) AV250 strain and classical swine fever virus (CSF) AV65 strain.

The European PRRSV SE strain was obtained from the European Triangle Research Centre in Hungary, and the European PRRSV PL strain was obtained from the OIE Reference Laboratory of Porcine Reproductive and Respiratory Syndrome of the National Veterinary Research Institute in Poland. RNA samples were provided.

2. Clinical samples

The field samples used for clinical testing came from pig farms in different areas of Gansu Province, with a total of 596 samples, including 129 pig serum and 467 tissue samples. Of the 129 pig sera, 52 were positive and 77 were negative. Tissue samples included 82 lung tissues (43 positive, 39 negative), 68 liver tissues (36 positive, 32 negative), 68 heart tissues (33 positive, 35 negative), and 88 kidney tissues ( 47 samples were positive and 41 samples were negative), 71 samples of spleen tissue (35 samples were positive, 36 samples were negative), and 90 samples were lymph nodes (45 samples were positive and 45 samples were negative). Rapid chromogenic one-step RT-LAMP and conventional RT-PCR assays for clinical specificity and sensitivity of the newly established method.

3. Nucleic acid amplification and extraction related kits

The amplification kits used to establish the RT-LAMP method were RT-LAMP kits from Beijing Meilaibo Medical Technology Co., Ltd. and Loopamp Fluorescence Visual Detection Kits (containing calcein and manganese ions) from Japan RIKEN Company. Nucleic acid extraction was performed using TaKaRa MiniBEST Viral RNA/DNAExtraction Kit Ver.5.0 produced by Takara Bioengineering (Dalian) Co., Ltd. The kit for conventional PCR amplification was derived from the Premix Taq Version 2.0 plus dye from Takara Bioengineering (Dalian) Co., Ltd. The kit used for RT-PCR is Takara's PrimeScriptTM One Step RT-PCR Kit Ver.2.

Example 1

1. Sample nucleic acid extraction

The MiniBEST Viral RNA/DNA Extraction Kit produced by Takara Bioengineering (Dalian) Co., Ltd. was used to extract nucleic acids from cytotoxic, serum and ground tissue samples.

2. Determination of NA-PRRSV by conventional RT-PCR reaction experiments

1. Primers used in RT-PCR reaction

Table 1 NA-PRRSV conventional RT-PCR amplification primer sequences

P224/P225 is the outer primer pair of the classical RT-LAMP method established by the applicant in 2008 (Chen et al. 2008), and the expected amplification fragment size for NA-HP-PRRSV is 246bp, and for NA-Non-HP-PRRSV amplification The predicted size of the augmented fragment is 336 bp.

2. RT-PCR amplification reaction system

RT-PCR amplification was performed using Takara's PrimeScriptTM One Step RT-PCR Kit Ver.2.

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 1μl

Add DEPC water to 20 μl.

3. RT-PCR amplification reaction process

Amplification program: 50℃, 30min; 94℃, 5min; (94℃, 30s; 58℃, 30s; 72℃, 40s/90s; 36 cycles); 72℃, 10min.

4. RT-PCR amplification product electrophoresis detection

RT-PCR amplification products were detected by conventional agarose gel electrophoresis, the gel concentration was 1%, the running buffer was 1xTAE, and the electrophoresis speed was 5-6 volts/cm (ie, voltage drop).

3. NA-HP-PRRSV rapid color development one-step RT-LAMP establishment

1. NA-HP-PRRSV rapid color development one-step RT-LAMP amplification primer selection and sequence

The applicant analyzed and compared the whole genome sequences of the American type highly pathogenic PRRSV strain, the American type non-highly pathogenic PRRSV strain and the European type PRRSV strain, using DNAstar software and alignment, around the NA-HP- Specific primers were designed in the 90-base deletion region of the NSP2 non-structural coding gene of PRRSV, aiming to distinguish the above three types of PRRSV.

NA-HP-PRRSV rapid color development one-step RT-LAMP has a total of 6 primers (see Table 2), in addition to the conventional pair of outer primers (F3 and B3) and a pair of inner primers (FIP and BIP), there is also a pair of primers Loop primers, namely FLooP and BLooP. The purpose of introducing the loop primer is to further improve the amplification efficiency, increase the product quantity, and facilitate rapid color development.

Table 2 HP-PRRSV rapid color development one-step LAMP primer sequences

2. RT-LAMP amplification reaction system

The reaction system of NA-HP-PRRSV RT-LAMP is 25μL/tube, and the sample volume is as follows:

PM Premix Primer: FIP/BIP (50UM) 0.4 μL

FLOOP/BLOOP (50UM) 0.4 μL

F3/B3 (10UM 0.2 μL

Extracted RNA: 1.0-2.0 μL

Bst enzyme 1.5 μL

2×U-load mix buffer: 12.5 μL

Eiken Loopamp Fluorescent Dye (Contains Calcein and Manganese) 0.4-0.5 μL

DEPC-treated ddH ₂ O supplemented to 25.0 μL

Total volume 25.0 μL.

The RT-LAMP reaction involves 6 primers. Before the experiment, the 6 primers were pre-mixed according to the amount to prepare the pre-mixed primers. During the operation, it was calculated according to (1 μL of the pre-mixed primers)/tube. The amount of the calcin-containing fluorescent dye can be added to 0.4-0.5 μL/25 μL reaction system/tube, which reduces the cost on the one hand, and reduces the inhibition of the amplification reaction by the calcein-containing fluorescent dye on the other hand.

3. RT-LAMP amplification reaction process

RT-LAMP is a constant temperature amplification reaction. Therefore, PCR amplification instruments are not required, as long as the instruments that can provide constant temperature reactions are available. The most commonly used instruments are ordinary water baths or heat blocks. After adding the sample, the reaction tube was centrifuged for an instant, and then placed in a water bath (the tube can be inserted into the float) or a heat block.

Amplification procedure: 60-65°C reaction for 45-90 minutes. Usually 60 minutes is enough. 63 ℃ or 64 ℃ reaction is the best.

4. RT-LAMP amplification product electrophoresis detection

(1) Agarose gel electrophoresis detection method

RT-LAMP amplification products can be detected by conventional agarose gel electrophoresis, the gel concentration is 2-2.5%, the running buffer is 1×TAE, and the electrophoresis speed is 5-6 volts/cm (ie voltage drop). After electrophoresis, the gel was placed in a stain containing EB for 30 minutes, then observed under UV light and photographed by computer.

(2) Fluorescence visual inspection method

Rapid color development of one-step RT-LAMP is due to the addition of dyes containing calcein and manganese ions into the reaction tube, so after the reaction, the positive sample tube will show bright green fluorescence, while the control tube and negative sample tube only have Faint background fluorescence. In this way, by comparing with the negative and positive controls set by the system, the judgment can be made by visual observation under natural light. Furthermore, since the positive sample produced a large amount of product due to the amplification, the solution of the positive tube was more turbid than that of the negative control. In the laboratory, the color change can also be observed under ultraviolet light, which is more obvious than natural light.

Fourth, the performance test of the method of the present invention

1. Sensitivity test of NA-HP-PRRSV rapid color development one-step RT-LAMP analysis

RNA was extracted from the Mark-145 cytotoxicity of NA-NA-HP-PRRSV QH08 strain, and the nucleic acid concentration was measured by UV spectrophotometer, and then the amount of extracted RNA per microliter was calculated based on the molecular weight of the nucleic acid of the whole genome (Genebank accession number KT033733). Nucleic acid copy number. The RNA samples were serially diluted with sterile and nuclease-free ddH ₂ O to 1×10^6-1×10^0 (nucleic acid copy number)/μL, and then RT-LAMP experiments were performed as described above, each reaction tube Add 1 μL of the above serially diluted RNA sample as template. After the reaction, agarose gel electrophoresis and fluorescence detection were performed. At the same time, the above-mentioned double-diluted RNA samples were amplified by conventional RT-PCR, and the analytical sensitivity of the two methods was compared.

2. NA-HP-PRRSV rapid color development one-step RT-LAMP analysis specificity experiment

To test the analytical specificity of this method, cross-reactivity experiments were performed with other common viruses that can cause reproductive disorders in pigs, such as NA-Non-PRRSV, EU-PRRSV, PCV1, PCV2, PPV1, PRV and CSF. . The specific strains are PCV1 (CAU0672), PCV2 (CAU0673), PPV1 type AV30 strain and AV31 strain, PRV AV250 strain, CSF AV65 strain and CH-1R strain of NA-Non-HP-PRRSV. The European PRRSV SE strain was from the European Triangle Research Center in Hungary, and the European PRRSV PL strain was from the OIE Reference Laboratory of Porcine Reproductive and Respiratory Syndrome, National Veterinary Research Institute, Poland, both of which were RNA samples.

3. NA-HP-PRRSV rapid color development one-step RT-LAMP clinical sensitivity test

The positive samples used for NA-HP-PRRSV rapid display one-step RT-LAMP clinical sensitivity analysis were obtained from tissue and serum samples from pig farms in different areas of Gansu province. There were 52 serum samples, 43 lung tissue samples, 36 liver tissue samples, 33 heart tissue samples, 47 kidney tissue samples, 35 spleen tissue samples, and 45 lymph node samples, totaling 291 samples. These samples were identified as NA-HP-PRRSV infection by comprehensive analysis methods such as ELISA and cell inoculation. After RNA extraction from these samples, NA-HP-PRRSV rapid display one-step RT-LAMP and conventional RT-PCR assays were performed, respectively.

4. NA-HP-PRRSV rapid color development one-step RT-LAMP clinical specificity test

The negative samples used for NA-HP-PRRSV rapid chromogenic one-step RT-LAMP clinical specific analysis were obtained from healthy pig farms in different regions of Gansu Province, with a total of 305 samples, including 77 serum samples and 228 tissue samples. The tissue samples included 39 lung tissues, 32 liver tissues, 35 heart tissues, 41 kidney tissues, 36 spleen tissues, and 45 lymph nodes. After nucleic acid extraction from these samples, NA-HP-PRRSV rapid chromogenic one-step RT-LAMP and conventional RT-PCR assays were performed to evaluate the clinical specificity of the newly established rapid chromogenic one-step RT-LAMP.

【result】

1. Conventional RT-PCR method for detection of NA-HP-PRRSV

The P224/P225 primer pair designed around the 90-base deletion region of the NA-HP-PRRSV non-structural protein NSP2 encoding gene was used to target the NA-HP-PRRSV strain QH08, Non-HA-PRRSV strain CH-1R and RNA template amplification of EU-PRRSV strain SE, the results are shown in Figure 1. The electrophoresis map shows that the P224/P225 primers are used to amplify and do not cross-react with EU-PRRSV. The size of the amplified fragment for NA-HP-PRRSV is expected to be 246 bp, and the size of the amplified fragment for NA-Non-HP-PRRSV is expected to be 336 bp. .

Figure 1 is an agarose gel electrophoresis map of PRRSV amplified by RT-PCR (P224/P225 primers); among them, M: DL2000 molecular marker (2000, 1000, 750, 500, 250, 100 bp); 1. RNA of EU-PRRSV strain SE; 2. NA-NA-HP-PRRSV QH08 cytotoxic RNA; 3. NA-Non-HP-PRRSVCH-1R cytotoxic RNA.

2. NA-HP-PRRSV rapid color development one-step RT-LAMP establishment

Using the cytotoxic RNA of NA-NA-HP-PRRSV QH08 strain as a template, the NA-HP-PRRSV rapid color development one-step RT-LAMP was established. Bands, the green fluorescence of the reaction tubes are all bright in the UV mode of the UV light of the gel imager. At 63 and 64 °C, the bands were more distinct, and the fluorescent color of the reaction tube was stronger and brighter. Under natural light conditions, the green fluorescence is visible to the naked eye as a function of temperature. The photo effect is not as good as the naked eye observation, as shown in Figure 2. It can be seen from the results in Figure 2 that the suitable amplification temperature of the one-step RT-LAMP for rapid color development of HP-PRRSV is 63°C and 64°C.

Figure 2 shows the RT-LAMP spectrum of HP-PRRSV rapid color development one-step method under different temperature conditions at 60-65 °C;

Panel A: Agarose gel electrophoresis pattern of LAMP amplification products;

Figure B: The corresponding reaction tube of the LAMP amplification product was photographed under the UV mode of the UV light of the gel imager;

Picture C: The corresponding reaction tube of the LAMP amplification product was photographed in the black and white mode of the UV light of the gel imager;

Figure D: The corresponding reaction tube of the LAMP amplification product was photographed under natural light conditions;

M: DL2000 Marker (2000, 1000, 750, 500, 250, 100 bp); 1, 2, 3, 4, 5, 6 represent isothermal amplification at 60, 61, 62, 63, 64, and 65°C, respectively.

3. Analytical Sensitivity Detection of NA-HP-PRRSV Rapid Chromogenic One-Step RT-LAMP

The whole gene of NA-NA-HP-PRRSV QH08 is 15349bp in length. According to ssRNA, the average molecular weight of each base is 340 Daltons, and its average molecular weight is 5218660 Daltons. When its RNA concentration is 1ng/μl, it contains gene copies The number is 1.15x10^8/μl. After 10-fold serial dilution, rapid color development one-step LAMP (isothermal amplification at 65°C) and conventional RT-PCR were performed, and the sensitivities of the two were compared.

The results showed that when the RNA nucleic acid extracted from cytotoxicity was used as the detection object, the detection limit of the rapid color development one-step LAMP was 10 copies/μL (Figure 3), and the detection limit of the conventional RT-PCR method (P224/P225 primer pair) The out-limit was around 1x10^6 copies/μL (Fig. 4A). Using the purified product amplified by P224/P225 (amplified with QH08 RNA as template) as template, 10-fold serial dilution was carried out according to 1-10^8, the detection limit of conventional RT-PCR method was 100 copies/ μL (Fig. 4B), while the LAMP method remained at 10 copies/μL (refer to Fig. 3, as the graphical bands did not change). This shows that the one-step rapid color development RT-LAMP is more sensitive than conventional RT-PCR, which can be improved by about 10 times (using purified DNA fragments as templates) and 100,000 times (using RNA as templates).

Figure 3 shows the results of a 10-fold serial dilution of NA-HP-PRRSV QH08 strain RNA as a template, and the results of one-step RT-LAMP detection of HP-PRRSV rapid color development;

Panel A: Agarose gel electrophoresis pattern of RT-LAMP amplification products;

Figure B: The corresponding reaction tube of the RT-LAMP amplification product was photographed under the UV mode of the UV light of the gel imager;

Picture C: The corresponding reaction tube of the RT-LAMP amplification product was photographed in the black and white mode of the UV light of the gel imager;

M: DL2000 Marker (2000, 1000, 750, 500, 250, 100bp); the corresponding template copy numbers (copy number/μL) are 1(0); 2 (1×10^0); 3 (1×10^1); 4 (1×10^2); 5 (1×10^3); 6 (1×10^4); 7 (1×10^5); 8 (1×10^6); 9 (1×10^) 7);10(1×10^8).

Figure 4 shows the analytical sensitivity detection of RT-PCR (P224/P225 primers) with 10-fold serial dilution of NA-HP-PRRSV QH08 strain RNA as template (4A) and purified DNA fragment as template (4B); ,

Picture A is using RNA as template, and picture B is using purified DNA fragment as template.

M: DL2000 Marker; the number of RNA template copies in lanes 1-10 is 0-1x10^8.

4. Analysis specificity of NA-HP-PRRSV rapid chromogenic one-step RT-LAMP

The newly established NA-HP-PRRSV rapid chromogenic one-step RT-LAMP has strong specificity and reacts only with NA-HP-PRRSV reference strains (TJM-F92, HUN4-F112, JXA1-R, HPBEDV and LNXM) , not with NA-Non-HP-PRRSV reference strains (CH-1R and CH-1a), EU-PRRSV reference strains (SE and PL) and PCV1 (CAU0672), PCV2 (CAU0673), PPV1 type AV30 strains ( CVCCAV30), PRV (CVCCAV250) and CSF (CVCCAV65) cross-reacted (see Figure 5).

Figure 5 is the analysis specificity detection result of NA-HP-PRRSV rapid color development one-step RT-LAMP; wherein,

Panel A: Agarose gel electrophoresis pattern of RT-LAMP isothermal amplification products;

Picture B: The corresponding reaction tube of the RT-LAMP isothermal amplification product was photographed in the black and white mode of the UV light of the gel imager;

Picture C: The corresponding reaction tube of RT-LAMP isothermal amplification product was photographed under the UV mode of UV light of the gel imager;

M: DL2000 Marker (2000, 1000, 750, 500, 250, 100 bp); using nucleic acids of different viruses as templates, in order: (1) ddH ₂ O; (2) NA-NA-HP-PRRSV QH08 strain; (3) NA-HP -PRRSV TJM-F92 strain; (4) NA-HP-PRSSV HUN4-F112 strain; (5) NA-HP-PRRSV JXA1-R strain; (6) NA-HP-PRRSV HPBEDV strain; (7) NA-HP - PRRSV LNXM strain; (8) Non-HP-PRRSV CH-1R strain; (9) Non-HP-PRRSV CH-1a strain; (10) PPV1 type AV30 strain (CVCCAV30); (11) PCV1 (CAU0672); (12) PCV2 (CAU0673); (13) PRV (CVCC AV250); (14) Kit provides positive control; (15) CSF (CVCCAV65); (16) EU-PRRSV SE strain; (17) EU-PRRSV PL strains.

5. Clinical Sensitivity Detection of NA-HP-PRRSV Rapid Chromogenic One-Step RT-LAMP

291 HP-PRRSV positive clinical samples were used to detect the clinical sensitivity of NA-HP-PRRSV rapid chromogenic one-step RT-LAMP, and compared with conventional RT-PCR. The results in Table 3 show that the overall positive detection rate of the RT-LAMP method was 96.2%. The total detection rate of conventional RT-PCR amplified with primer pair P224/P225 was 90.4%. For pig serum, the detection rate of RT-LAMP method was 96.2%, and the detection rate of RT-PCR method was 90.4%. For tissue samples, the detection rate of RT-LAMP was 90.9-100% and that of RT-PCR was 84.8-95.3%. The above results show that the newly established NA-HP-PRRSV rapidly shows that the clinical sensitivity of one-step RT-LAMP is significantly higher than that of RT-PCR, and it has a high detection rate for serum samples, which is suitable for clinical biopsy.

Table 3 Sensitivity comparison of rapid chromogenic one-step RT-LAMP and RT-PCR detection in 291 NA-HP-PRRSV positive clinical samples

6. Clinical specific detection of NA-HP-PRRSV rapid chromogenic one-step RT-LAMP

The clinical specific analysis of RT-LAMP was performed on samples collected from farms without porcine reproductive disorders and compared with RT-PCR. A total of 305 samples were tested, including 77 serum samples and 228 tissue samples. For these negative clinical samples, the overall detection rate of one-step RT-LAMP was 95.4% and that of conventional RT-PCR was 93.1%. The detection rates for serum samples were 97.4% (RT-LAMP) and 93.1% (RT-PCR). The detection rate of RT-LAMP in tissue samples was 93.4%-97.4%, and the detection rate of RT-PCR was (90.6-94.8%). Both methods have good specificity, but the rapid chromogenic one-step RT-LAMP method is somewhat more specific than the RT-PCR method. Lungs, lymph nodes and blood are suitable samples for RT-LAMP and RT-PCR assays. The newly established NA-HP-PRRSV rapid chromogenic one-step LAMP method has strong specificity and can detect various tissue samples including serum, so it is suitable for clinical application.

Table 4 Comparison of specificity between NA-HP-PRRSV rapid chromogenic one-step RT-LAMP and RT-PCR methods in 267 negative clinical samples

Finally, it should be noted that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, the The technical solutions described in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (6)

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. 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.