Fluorescent PCR (Polymerase Chain Reaction) amplification primer probe group and detection kit capable of detecting porcine tokoro virus type 1

Abstract

The invention relates to the technical field of pathogen detection, in particular to a fluorescent PCR (Polymerase Chain Reaction) amplification primer probe group and a detection kit capable of detecting porcine tokeno virus type 1. The primer probe group comprises a primer pair and a probe, the primer pair comprises an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2, and the probe comprises a probe and a probe. And the probe comprises a probe as shown in SEQ ID NO. 3. The primer probe group disclosed by the invention can be used for effectively detecting the porcine tokeno virus type 1, and comprises a primer combination and a probe which are high in specificity and sensitivity. The kit disclosed by the invention is sensitive, convenient, high in specificity, high in sensitivity and good in reliability, and whether a sample contains the porcine Tokinovirus type 1 or not can be judged only through one-time detection.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen detection, and particularly to a fluorescence PCR amplification primer-probe set and a detection kit for detecting TorqueTenoSus Virus Type 1 in pigs. Background Art

[0002] TorqueTenoSus Virus Type 1 (TTSuV-1) is a circular single-stranded DNA virus, also known as porcine circovirus type 1. This virus is widely present in primary and subculture pig-derived cells of different passages and in the bodies of normal pigs. It mainly causes symptoms such as fever, listlessness, rough hair, progressive weight loss, anemia, muscle atrophy, weakness, dyspnea, cough, watery diarrhea or black stools, causing a certain degree of harm to the pig breeding industry.

[0003] The existing patent application with the publication number CN109852729A and the invention name of a real-time fluorescence quantitative detection kit for porcine circovirus type k2b, although the primers and probes used have strong specificity and high sensitivity, it can only detect porcine circovirus type k2b and cannot detect TorqueTenoSus Virus Type 1.

[0004] Currently, traditional detection methods such as virus isolation and culture are time-consuming and laborious, and ELISA kits also have problems such as poor sensitivity and insensitivity in detecting at the early stage of virus infection. Therefore, a rapid and sensitive detection method is of great significance for the detection and prevention of TorqueTenoSus Virus Type 1. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a fluorescence PCR amplification primer-probe set and a detection kit for detecting TorqueTenoSus Virus Type 1 in pigs, so as to rapidly and sensitively detect and prevent TorqueTenoSus Virus Type 1.

[0006] Based on the above purpose, the present invention provides a fluorescence PCR amplification primer-probe set for detecting TorqueTenoSus Virus Type 1 in pigs. The primer-probe set includes a primer pair and a probe. The primer pair includes an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, and the probe includes a probe shown in SEQ ID NO.3.

[0007] The present invention also provides a fluorescence PCR detection kit for detecting TorqueTenoSus Virus Type 1 in pigs, and the kit includes the fluorescence PCR amplification primer-probe set for detecting TorqueTenoSus Virus Type 1 in pigs.

[0008] The kit further includes 2×Probe qPCR MasterMix and a fluorescence PCR template diluent.

[0009] The kit also includes a porcine torovirus 1 type qPCR positive control and ultrapure water. To increase product stability and avoid the spread of infectious pathogens, live samples are not provided as positive controls in this product. Only non-infectious DNA fragments are provided as positive controls. The positive control is 1E7 copies / μL and is used for standard curve construction and identification of false negative samples. Preferably, the porcine torovirus 1 type qPCR positive control is an artificially synthesized DNA sequence.

[0010] The PCR reaction system used in the kit is 20 μL, which consists of 10 μL of 2×Probe qPCR MasterMix, 3 μL of the probe primer mixture, and 7 μL of the DNA template to be tested.

[0011] Preferably, the amplification reaction program adopted by the kit is 95 °C for 90 sec; 40 cycles, each cycle being 95 °C for 15 sec and 55.4 °C for 60 sec; the fluorescence signal is collected in the second step of each cycle.

[0012] When detecting with the kit, when the Ct value ≤ 40 and a specific amplification curve appears in the channel corresponding to the probe, it is determined that porcine torovirus 1 type is contained.

[0013] In the present invention, the primers and probes are designed based on the 5' UTR region gene of porcine torovirus 1 type.

[0014] Advantages of the present invention: The primer-probe group of the present invention can effectively detect porcine torovirus 1 type, and it includes a primer combination and a probe with strong specificity and high sensitivity. The kit of the present invention is sensitive, convenient, highly specific, sensitive, and reliable. It can determine whether a sample contains porcine torovirus 1 type with just one detection, detect the content of porcine torovirus 1 type, and can also analyze a large number of samples simultaneously, providing strong technical support for the qualitative / quantitative detection of porcine torovirus 1 type, and has great practical value and good application prospects. Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1 It is a schematic diagram of the primer-probe design of the present invention;

[0017] Figure 2 It is a schematic diagram of the feasibility verification of the primer-probe of the present invention;

[0018] Figure 3 Schematic diagram for sensitivity verification of the primer-probe of the present invention. Detailed implementation manners

[0019] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific embodiments.

[0020] It should be noted that unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those with ordinary skills in the field to which the present invention belongs. Words such as "including" or "comprising" and the like mean that the elements or objects appearing before this word cover the elements or objects listed after this word and their equivalents, without excluding other elements or objects.

[0021] Example 1

[0022] Design of a fluorescence PCR amplification primer and probe for detecting Porcine Torque teno virus type 1.

[0023] Download the genomic sequence of the 5' UTR region of Porcine Torque teno virus type 1 from NCBI. To ensure the detection effect and improve the detection rate of Porcine Torque teno virus type 1, primers and probes are designed according to the highly conserved region 5' UTR of Porcine Torque teno virus type 1 (the primer and probe sequences are shown in Table 1, and the schematic diagram is as Figure 1 shown). Among them: F represents the upstream primer; P represents the probe; R represents the downstream primer.

[0024] Table 1 Design of fluorescence PCR primers / probes

[0025]

[0026]

[0027] Feasibility and sensitivity verification of the above fluorescence PCR amplification primer and probe for detecting Porcine Torque teno virus type 1.

[0028] First, dilute the standard curve samples. Using the provided positive control with a concentration of 1E7 / μL as the stock solution, perform 10-fold serial dilutions with the dedicated template diluent for fluorescence PCR to obtain six standard curve samples with dilution gradients of 1E1 - 1E6 copies / μL. Use the Q1600 real-time fluorescence quantitative PCR instrument from Hangzhou Baiheng Technology Co., Ltd. for detection. The total reaction system for a single detection is 20 μL. Among them, the components of the six standard curve sample tubes include 10 μL of 2×Probe qPCR MasterMix each, 3 μL of the qPCR primer-probe mixture for porcine torovirus type 1 each, and 7 μL of the standard curve sample diluent each; the components of the PCR negative control include 10 μL of 2×Probe qPCR MasterMix each, 3 μL of the qPCR primer-probe mixture for porcine torovirus type 1 each, and 7 μL of ultrapure water. The reaction program is in a fixed mode, i.e., pre-denaturation at 95°C for 90 sec; 40 cycles, with each cycle being 95°C for 15 sec and 55.4°C for 60 sec; collect fluorescence signals in the second step of each cycle.

[0029] The obtained data is shown in Table 2. Construct a standard curve with the log value of the positive control concentration on the horizontal axis and the Ct value on the vertical axis and perform linear fitting.

[0030] According to the results ( Figure 2 and Figure 3 ), it can be seen that the primers and probes have good performance and high sensitivity. The lowest detection limit detected with the positive control is not less than 100 copies / reaction, the linear relationship is good, and the fitting degree R 2 is not less than 0.95. The linear range during quantitative detection can reach at least six orders of magnitude (where numbers ① - ⑥ correspond to the positive control concentrations of E6 - E1).

[0031] Table 2 Ct values corresponding to positive controls with different concentrations

[0032] E1 E2 E3 E4 E5 E6 NC 33.65 30.4 26.53 22.71 18.89 15.69 Undetermined

[0033] Specificity verification of the fluorescence PCR amplification primers and probes for detecting porcine torovirus type 1

[0034] Design to amplify positive samples of porcine parvovirus, porcine circovirus, and porcine teschovirus using the fluorescence PCR method established in the present invention, and the results are all negative.

[0035] Repeatability verification of the fluorescence PCR amplification primer-probe set and detection kit for detecting porcine torovirus type 1

[0036] Plasmids with known concentrations (1E6 copies, 1E4 copies, 1E2 copies) were used as templates, and an operator repeated the detection on the same day to verify the intra-group repeatability of the method. Two operators performed the operations at different times to calculate the inter-group repeatability. As shown in Table 3 below, the results showed that the intra-group and inter-group repeatability of the method was good, and the coefficients of variation were 2.85% - 3.45% and 3.25% - 4.11% respectively.

[0037] Table 3: Verification of the repeatability of the fluorescence PCR method

[0038]

[0039] Detection of different types of samples using the above fluorescence PCR amplification primer-probe set and detection kit capable of detecting Porcine Torque teno virus type 1

[0040] Taking DNA of different types of positive samples for detection as shown in Table 4 below, the results showed that all detections using the method of the present invention were positive.

[0041] Table 4: Verification of the detection method of the present invention for different types of positive DNA

[0042]

[0043] Those of ordinary skill in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the present invention is limited to these examples; under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, which are not provided in detail for the sake of brevity. Any omission, 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

1. A fluorescence PCR amplification primer-probe set for detecting porcine torovirus type 1, characterized in that, The primer-probe set includes a primer pair and a probe. The primer pair includes an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, and the probe includes a probe shown in SEQ ID NO.

3.

2. A fluorescence PCR detection kit for detecting porcine torovirus type 1, characterized in that, The kit includes the fluorescence PCR amplification primer-probe set for detecting Porcine Torque teno virus type 1 as described in claim 1.

3. The fluorescence PCR detection kit for detecting porcine torovirus type 1 according to claim 2, characterized in that, The kit further includes 2×Probe qPCR MasterMix and a fluorescence PCR template diluent.

4. The fluorescence PCR detection kit for detecting porcine Torque teno virus type 1 according to claim 2, wherein The kit further includes a Porcine Torque teno virus type 1 qPCR positive control and ultrapure water.

5. The fluorescent PCR detection kit for detecting porcine torque teno virus type 1 according to claim 4, wherein The Porcine Torque teno virus type 1 qPCR positive control is an artificially synthesized DNA sequence.

6. The fluorescence PCR detection kit for detecting porcine torque teno virus type 1 according to claim 2, wherein, The amplification reaction program adopted by the kit is 95°C for 90 sec; 40 cycles, each cycle being 95°C for 15 sec and 55.4°C for 60 sec; the fluorescence signal is collected in the second step of each cycle.

7. The fluorescence PCR detection kit for detecting porcine torovirus type 1 according to claim 2, wherein When detecting with the kit, when the Ct value ≤ 40 and a specific amplification curve appears in the channel corresponding to the probe, it is determined that Porcine Torque teno virus type 1 is contained.

Images