CN105177179A - RT-LAMP primer group and kit for detecting porcine epidemic diarrhea virus and application thereof - Google Patents

Abstract

The invention discloses an RT-LAMP primer set, kit and application thereof for detecting porcine epidemic diarrhea virus. The primer set is composed of a pair of outer primers, a pair of inner primers and a pair of loop primers, the outer primer pair sequences are shown in SEQIDNo.1 and SEQIDNo.2, and the inner primer pair sequences are SEQIDNo.3 and SEQIDNo. As shown in 4, the sequence of the loop primer pair is SEQIDNo.5 and SEQIDNo.6, which can effectively detect porcine epidemic diarrhea virus. The kit includes the aforementioned primers and can be used for detecting porcine epidemic diarrhea virus. The invention also provides a method for detecting porcine epidemic diarrhea virus by using the aforementioned kit, the detection result can be intuitively judged according to the color change of the reaction solution, and is fast and accurate. The invention has the characteristics of strong specificity, high sensitivity, fast and accurate detection of porcine epidemic diarrhea virus, and is especially suitable for rapid detection on the spot.

Description

RT-LAMP primer set, kit and application for porcine epidemic diarrhea virus detection

technical field

The invention relates to the technical field of detection of porcine epidemic diarrhea virus.

Background technique

Porcine epidemic diarrhea (porcineepidemicdiarrheavirus, PED) is caused by porcine epidemic diarrhea virus (porcineepidemicdiarrheavirus, PEDV), a highly contagious disease of pigs with diarrhea, vomiting and dehydration as the main clinical features. It first appeared in the UK in 1971, and the disease occurred successively in my country in the early 1980s. At present, the disease has been reported in many major pig-raising countries in the world.

PEDV detection methods mainly include: virus isolation, RT-PCR, real-time fluorescence quantitative PCR. These methods have played an important role in virus detection, but they generally have disadvantages such as cumbersome operation, long detection cycle, and expensive equipment, and are not suitable for rapid detection at the grassroots level or on-site.

The reverse transcription loop-mediated isothermal amplification technology (ReverseTranscriptionLoop-Mediated Isothermal Amplification, RT-LAMP) is specially used for rapid amplification of RNA. The basic principle is to use 4 or 6 specific primers and a DNA polymerase with strand displacement function to amplify nucleic acid under isothermal conditions. The test cycle is very short and the reaction can be completed within one hour. After the reaction is completed, you only need to observe the result through its by-product white magnesium pyrophosphate precipitation, or add a fluorescent dye in advance, and the result can be judged by visual observation.

The RT-LAMP method has the advantages of simplicity, rapidity, sensitivity, and specificity, and is especially suitable for rapid detection at the grassroots level or on the spot. In RT-LAMP technology, primers are the key factors that determine the sensitivity and specificity of detection results. At present, it is urgent to establish a RT-LAMP detection method that can detect porcine epidemic diarrhea virus sensitively, specifically and efficiently.

Contents of the invention

To solve the above problems, the present invention designs a set of general RT-LAMP-specific primers in the conserved sequence region of its N gene according to the porcine epidemic diarrhea virus sequence published by GenBank, as shown in Table 1. The RT-LAMP established with the primers was used to detect porcine transmissible gastroenteritis virus, rotavirus and porcine reproductive and respiratory syndrome virus and other three porcine disease pathogens. The results showed that the primer set could only specifically amplify porcine epidemic Diarrhea virus genes, while not able to amplify other viral genes, have good specificity.

One of the technical problems solved by the present invention is to provide a RT-LAMP primer set for porcine epidemic diarrhea virus detection, characterized in that: the primer set consists of a pair of outer primers, a pair of inner primers and a pair of loops Primer composition; the outer primer pair sequence is shown in SEQIDNo.1 and SEQIDNo.2, the inner primer pair sequence is shown in SEQIDNo.3 and SEQIDNo.4, and the loop primer pair sequence is SEQIDNo.5 and SEQIDNo. 6.

The primer sequences corresponding to SEQIDNo.1, SEQIDNo.2, SEQIDNo.3, SEQIDNo.4, SEQIDNo.5, SEQIDNo.6 were named as N-F3, N-B3, N-FIP, N-BIP, N- LF, N-LB.

Specifically, the molar ratio of the primers N-F3, N-B3, N-FIP, N-BIP, N-LF, and N-LB can be 1:1:4:4:2:2.

In one embodiment of the present invention, the final concentration of the pair of outer primers (N-F3 and N-B3) in the RT-LAMP reaction system is 0.2 μM, and the pair of inner primers (N-FIP and N -BIP) in the RT-LAMP reaction system had a final concentration of 0.8 μM, and the pair of loop primers (N-LF and N-LB) had a final concentration of 0.4 μM in the RT-LAMP reaction system.

The second technical problem solved by the present invention is to provide a kind of RT-LAMP kit for porcine epidemic diarrhea virus detection, comprising RT-LAMP reaction liquid, strand displacement DNA polymerase and reverse transcriptase, its characteristic In that it also includes the above-mentioned primer set.

Preferably, the RT-LAMP reaction solution is composed of 10×BstDNABuffer, MgCl ₂ , dNTPs, and nuclease-free water; the strand displacement DNA polymerase is BstDNA polymerase; the reverse transcriptase is composed of AMV reverse transcriptase and RNase inhibitor composition.

Preferably, the kit also includes a fluorescent reagent, a positive control and a negative control.

Wherein, the positive control can be porcine epidemic diarrhea virus sample; the negative control can be sterilized ultrapure water.

Preferably, the fluorescent chromogenic agent can be an aqueous solution containing calcein and MnCl ₂ .

Preferably, the kit also includes reagents for extracting RNA: TRIzolLSReagent, chloroform, isopropanol, 75% ethanol, and nuclease-free water.

The application of the following 1) or 2) also belongs to the protection scope of the present invention:

1) The application of the primer set as described above in the preparation of the kit;

2) The application of the primer set or the kit in the preparation of products for detecting whether porcine epidemic diarrhea virus is contained in the sample to be tested.

The method for detecting whether porcine epidemic diarrhea virus is contained in the sample to be tested by using the primer set or the kit comprises the following steps:

1. Extraction of viral RNA

The RNA of porcine epidemic diarrhea virus was extracted by the guanidinium isothiocyanate-phenol-chloroform one-step method. Specific steps are as follows:

(1) Add 250 μL sample to 750 μL TRIzolLS Reagent, shake vigorously for 2 minutes, and place at room temperature for 5 minutes. Add 250 μL of chloroform, shake vigorously for 1 min, leave at room temperature for 5 min, centrifuge at 12,000 rpm at 4°C for 15 min, and transfer the upper aqueous phase to a new centrifuge tube.

(2) Add isopropanol equal to the volume of water, mix well, let stand at room temperature for 15 minutes, centrifuge at 12,000 rpm at 4°C for 15 minutes, pour off the supernatant, then add 800 μL of DEPC-treated 75% ethanol, and centrifuge at 12,000 rpm at 4°C 5min, discard the supernatant.

(3) Air-dry the precipitate naturally or dry it in a drying oven at 50°C, fully dissolve it with an appropriate amount of nuclease-free water, and immediately perform RT-LAMP or store it at -80°C for later use.

2. RT-LAMP reaction

Add 12 μL of RT-LAMP reaction solution, 7 μL of primer set, 2 μL of the above RNA template, 1.5 μL of reverse transcriptase, 1.5 μL of BstDNA polymerase, and 1 μL of fluorescent reagent to the reaction tube to make a 25 μL RT-LAMP reaction system, in which 7 μL of primer The group consisted of 0.5 μL of N-F3 at a concentration of 10 μM, 0.5 μL of N-B3 at a concentration of 10 μM, 2 μL of N-FIP at a concentration of 10 μM, 2 μL of N-BIP at a concentration of 10 μM, 1 μL of N-LF at a concentration of 10 μM, 1 μL Consists of N-LB at a concentration of 10 μM. After mixing, the reaction tube was placed in a water bath, kept at 62°C for 60 minutes, and then stopped at 80°C for 10 minutes.

3. Detection

(1) Direct observation: Observe the color change of the reaction solution immediately after the reaction is completed. The positive sample reaction solution turns green, and the negative sample reaction solution remains orange-yellow.

(2) Centrifugal test: The reaction tube is cloudy after the positive sample is reacted, and the reaction tube is clear after the negative sample is reacted. After centrifugation at 4000rpm for 5 minutes, white magnesium pyrophosphate precipitates can be seen in the positive sample reaction tube, and there is no precipitation in the negative sample reaction tube.

(3) Electrophoresis detection: RT-LAMP amplification products were checked by 1% agarose gel electrophoresis. A positive reaction presents a unique ladder-like band, while a negative reaction shows no band.

The present invention uses a fluorescent chromogenic agent as an indicator, and the detection result can be directly judged by visually observing the color of the reaction liquid, thereby avoiding the false positive result of the next experiment caused by environmental pollution caused by opening the reaction tube cover. The present invention only needs a temperature-controllable water bath to complete the whole reaction, and does not need an expensive thermal cycler. RT-LAMP has a fast amplification speed, and the result can be obtained within 60 minutes; the detection sensitivity is high, and a minimum of 0.7pg viral RNA can be detected.

The invention can detect the porcine epidemic diarrhea virus on the feces, intestinal tract and contents of diarrhea pigs, and has a wide range of detection objects and strong applicability.

According to the porcine epidemic diarrhea virus sequence published by GenBank, the present invention designs a set of general RT-LAMP specific primers in the conserved sequence region of its N gene, which is universal and representative. The RT-LAMP established with the primers was used to detect porcine transmissible gastroenteritis and other three porcine disease pathogens. The results showed that the primer set could only specifically amplify porcine epidemic diarrhea virus genes, but not other viral genes. Has very good specificity.

The invention has the advantages of strong specificity, high sensitivity, simple equipment and operation, and easy observation of results, and is especially suitable for rapid detection at the grassroots level and on the spot, and has broad application prospects.

Description of drawings

Fig. 1 is the specific detection result of porcine epidemic diarrhea virus RT-LAMP;

1: porcine epidemic diarrhea virus; 2: porcine transmissible gastroenteritis virus; 3: rotavirus; 4 porcine reproductive and respiratory syndrome virus; 5: nuclease-free water;

Fig. 2 is the detection result of porcine epidemic diarrhea virus RT-LAMP sensitivity;

A: amplification curve of fluorescence isothermal amplification instrument; B: direct observation;

1: 7.08×101 ng/μl 2: 7.08× ¹⁰⁰ ng/μl3: 7.08× ^10-1 ng/ ^μl4 : 7.08× ^10-2 ng/μl5: 7.08× ^10-3 ng/μl6: 7.08× ^10-4 ng/μl7: 7.08×10 ^-5 ng/μl8: nuclease-free water.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and examples. However, the invention is not limited to the examples given. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1 detects the best embodiment of the kit of porcine epidemic diarrhea virus with RT-LAMP method

The kit of this embodiment includes the following RT-LAMP primer sets, see Table 1. The kits are divided into A box and B box, and A box is an RNA extraction kit. The composition and storage conditions of the kit are shown in Table 2. Box B is a porcine epidemic diarrhea virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit. The kit composition and storage conditions are shown in Table 3.

Among them, the RT-LAMP reaction solution is composed of 50uL 10×BstDNABuffer, 50uL MgCl2 solution with a concentration of 25mM, 60uL dNTPs solution with a concentration of 2.5mM, and 120uL nuclease-free water.

The reverse transcriptase consists of 20 uL of AMV reverse transcriptase at a concentration of 200 U/μL and 10 uL of an RNase inhibitor at a concentration of 50 U/μL.

The fluorescent chromogen is an aqueous solution containing calcein and MnCl ₂ .

The positive control is porcine epidemic diarrhea virus sample, and the negative control is sterilized ultrapure water.

Primers are aqueous solution, consisting of 40 uL of N-BIP primer with a concentration of 10 μM, 40 uL of N-FIP primer with a concentration of 10 μM, 10 uL of M-F3 primer with a concentration of 10 μM, 10 uL of M-B3 primer with a concentration of 10 μM, and 20 uL with a concentration of 10 μM The N-LF primer was combined with 20 uL of the N-LB primer at a concentration of 10 μM.

Table 1 Porcine epidemic diarrhea virus RT-LAMP primers

Table 2: RNA extraction kit composition, volume and storage conditions serial number Kit composition Volume (mL) Storage Conditions 1 TRIzol LS Reagent 12.5 4°C 2 Chloroform 5 4°C 3 Isopropanol 10 4°C 4 75% ethanol 12.5 4°C 5 nuclease free water 0.2 4°C

Table 3: Composition, volume and storage conditions of porcine epidemic diarrhea virus RT-LAMP kit serial number Kit composition Volume (μL) Storage Conditions 1 RT-LAMP reaction solution 280 -20°C 2 Bst DNA polymerase 25 -20°C 3 reverse transcriptase 30 -20°C 4 Primer 140 -20°C 5 Fluorescent reagent 13 -20°C (protect from light) 6 positive control 750 -20°C 7 negative control 750 -20°C

Embodiment 2 non-diagnostic purpose detects the detection method of porcine epidemic diarrhea virus with RT-LAMP method

The detection method of this embodiment uses the kit of Embodiment 1 for detection. The detection method of this embodiment comprises the following steps:

1. Viral RNA extraction Porcine epidemic diarrhea virus RNA was extracted by one-step method of guanidine isothiocyanate-phenol-chloroform. The specific steps are as follows: (1) Denaturation. Add 750 μL TRIzolLS Reagent to 250 μL of the sample to be tested, the positive control and the negative control, shake vigorously for 2 minutes, and place at room temperature for 10 minutes. Add 250 μL of chloroform, shake vigorously for 15 s, leave at room temperature for 10 min, centrifuge at 12,000 g for 10 min at 4°C, and transfer the upper aqueous phase (about 500-600 μL) into a new centrifuge tube. Wherein, the samples to be tested can be porcine feces with diarrhea, intestinal tract and their contents.

(2) RNA precipitation and washing. Add an equal volume of isopropanol (about 500-600 μL) to the water phase, precipitate at -20°C for 30 minutes, centrifuge at 12,000 g at 4°C for 10 minutes, discard the supernatant, add 1 mL of DEPC-treated 75% ethanol and shake gently , centrifuge at 7,500 g for 5 min at 4°C.

(3) RNA dissolution and storage. After the precipitate was naturally air-dried, it was dissolved in 10 μL of nuclease-free water and the reverse transcription reaction was performed immediately.

2. RT-LAMP reaction

The sample to be tested, the positive control and the negative control RNA template extracted above were subjected to RT-LAMP reaction respectively. According to the numbering sequence in Table 4, add each component into the reaction tube in turn and mix well to prepare a 25 μL RT-LAMP reaction system. The reaction tube was placed in a water bath, kept at 62°C for 60 minutes, and then stopped at 80°C for 10 minutes.

Table 4 RT-LAMP reaction system

Observe the color change of the reaction solution immediately after the reaction is completed. The positive reaction solution turns green, and the negative reaction solution remains orange-yellow.

The specificity test of the RT-LAMP of embodiment 3 porcine epidemic diarrhea virus

According to the process of embodiment 2, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus (PRRSV), porcine transmissible gastroenteritis virus (TGEV), rotavirus (RV) are respectively carried out RT-LAMP reaction, detect Specificity of the RT-LAMP method. The results are shown in Figure 1, the nucleic acid of porcine epidemic diarrhea virus was amplified with a unique "S" curve, while the other three porcine disease nucleic acids were not amplified with an "S" curve. The above results show that the RT-LAMP detection method of the present invention can specifically amplify porcine epidemic diarrhea virus without cross-reaction with other porcine disease virus nucleic acids, and the RT-LAMP method of the present invention has good specificity.

The susceptibility test of the RT-LAMP of embodiment 4 porcine epidemic diarrhea virus

Measured by an ultra-micro spectrophotometer, the nucleic acid template with a total RNA concentration of 70 ng/μL of porcine epidemic diarrhea virus is taken for determination. RT-LAMP was used to detect the 10-fold diluted porcine epidemic diarrhea virus nucleic acid template.

The RT-LAMP reaction process of porcine epidemic diarrhea virus is the same as that in Example 2, and the results are shown in Figure 2. RT-LAMP can detect at least 0.7 pg of porcine epidemic diarrhea virus nucleic acid template.

Based on the experimental results of Examples 3 and 4, it can be seen that the present invention is sensitive, specific, convenient and quick, and is suitable for rapid detection and early screening of porcine epidemic diarrhea virus at the grassroots level or on the spot.

Claims (7)

1. a kind of RT-LAMP primer group that is used for porcine epidemic diarrhea virus detection is characterized in that: described primer group is made up of a pair of outer primers, a pair of inner primers and a pair of loop primers; Described outer primer pair sequence is As shown in SEQIDNo.1 and SEQIDNo.2, the sequence of the inner primer pair is shown in SEQIDNo.3 and SEQIDNo.4, and the sequence of the loop primer pair is shown in SEQIDNo.5 and SEQIDNo.6. 2. A RT-LAMP kit for porcine epidemic diarrhea virus detection, comprising RT-LAMP reaction solution, strand-displacing DNA polymerase and reverse transcriptase, characterized in that it also includes the primer described in claim 1 Group. 3. the RT-LAMP kit that is used for porcine epidemic diarrhea virus detection according to claim 2, is characterized in that described RT-LAMP reaction solution is made up of 10 * BstDNABuffer, MgCl ₂ , dNTPs, nuclease-free water; The strand displacement DNA polymerase is BstDNA polymerase; the reverse transcriptase is composed of AMV reverse transcriptase and RNase inhibitor. 4. The RT-LAMP kit for porcine epidemic diarrhea virus detection according to claim 2 or 3, characterized in that: the kit also includes a fluorescent chromogenic reagent, a positive control and a negative control. 5. The RT-LAMP kit for porcine epidemic diarrhea virus detection according to claim 4, characterized in that: the fluorescent chromogen is an aqueous solution containing calcein and MnCl ₂ . 6. The RT-LAMP kit for porcine epidemic diarrhea virus detection according to claim 2 or 3, characterized in that: said kit also includes reagents for extracting RNA: TRIzolLSReagent, chloroform, isopropanol, 75 % ethanol, nuclease-free water. 7. Application of 1) or 2) as follows: 1) The application of the primer set described in claim 1 in the preparation of the kit described in claim 2; 2) Application of the primer set according to claim 1 or the kit according to claim 2 in the preparation of products for detecting whether porcine epidemic diarrhea virus is contained in the sample to be tested.