Primer composition, micro-fluidic chip, kit and method for simultaneously detecting multiple pepper viruses

Abstract

The invention provides a primer composition, a micro-fluidic chip, a kit and a method for simultaneously detecting various pepper viruses. The primer composition comprises the following primer groups: a primer group for detecting pepper mild mottle virus, a primer group for detecting tomato brown wrinkled fruit virus, a primer group for detecting tomato mottle mosaic virus, a primer group for detecting phoenix fruit mosaic virus, a primer group for detecting phoenix fruit mosaic virus, and a primer group for detecting phoenix fruit mosaic virus. The invention discloses a primer group for detecting tomato spotted wilf virus and a primer group for detecting tomato ringspot virus. Based on the LAMP means and in combination with the microfluidic technology, synchronous detection of six related viruses of pepper can be achieved at most through single sample adding, and the method has higher specificity and sensitivity compared with a conventional LAMP technology.

Description

Technical Field

[0001] The invention belongs to the technical field of virus detection, and particularly relates to a primer composition, a microfluidic chip, a kit and a method for simultaneously detecting multiple pepper viruses. Background Art

[0002] Pepper (Capsicum annuum L.) is an annual or limited perennial plant of the genus Capsicum in the Solanaceae family. As one of the most important spice crops in the world, it has excellent phenotypic, horticultural, agronomic and biodiversity, and has extremely high commercial value. Viral diseases have long been one of the main factors restricting pepper production, seriously threatening pepper yield, quality and economic benefits.

[0003] Peppers can be infected by both DNA viruses and RNA viruses. Pepper viruses can overwinter on other hosts, and seeds can also carry viruses. Molecular biology detection is to detect viruses at the nucleic acid level. It has high sensitivity and strong specificity. It can overcome some shortcomings of serology and other detection methods. It is more suitable for large-scale sample detection and is currently the most promising virus detection technology. For example, RT-PCR technology, nucleic acid hybridization technology, double-stranded RNA (dsRNA) electrophoresis technology, loop-mediated isothermal amplification technology, etc. are increasingly widely used in virus detection.

[0004] Loop-mediated isothermal amplification (LAMP) is a new in vitro isothermal amplification technology for specific nucleic acid fragments invented by Japanese researchers Notomi et al. in 2000. Its characteristics are that 4 to 6 specific primers are designed for the 6 regions of the target gene, and under the action of strand displacement DNA polymerase (Bst DNA polymerase), isothermal amplification is performed at 60 to 65°C, and 10 9 ~10 10 It can amplify nucleic acid at a rate of 2 times, and has the characteristics of simple operation, strong specificity, and easy product detection.

[0005] Microfluidic chips provide a method for accurately controlling and processing trace samples, and have the characteristics of low cost, small size and high throughput. In addition, the chip design is flexible, the entire system is easy to operate and controllable; its sample volume is greatly reduced compared to microarray chips; the closed operating environment increases the anti-pollution ability of the detection; its performance of enhancing the efficiency of material transfer can shorten the reaction time and improve sensitivity.

[0006] Cucumber mosaic virus and tobacco mosaic virus are dominant species in most pepper producing areas in China, but bean wilt virus, pepper mild mottle virus and other viruses are gradually becoming important viral pathogens in China's main pepper producing areas, and the infection of potato viruses to peppers is also on the rise. Based on the current status of virus infection in Solanaceae plants, providing a simple, fast, efficient and low-cost virus detection method has become an urgent problem to be solved. Summary of the invention

[0007] The object of the present invention is to overcome the deficiencies in the prior art, and to provide a new primer combination and a microfluidic chip, a detection kit and a corresponding detection method comprising the primer combination. Thus, the present invention is based on the LAMP method, and in combination with microfluidic technology, a single target gene sequence is detected independently in multiple independent reaction pools, and the circuit design process and pressure drive of the microfluidic chip can also be used to drive the entire detection reaction to complete. Compared with the ordinary tubular LAMP, it has the advantages of detecting multiple target genes with a single sample addition, and has the characteristics of simple operation, rapidity, high efficiency, and low cost.

[0008] After a large number of experiments and studies, the inventors of the present invention selected pepper mild mottle virus (PMMoV, tobacco mosaic virus genus), tomato brown rugose fruit virus (ToBRFV, tobacco mosaic virus genus), tomato mottle mosaic virus (ToMMV, tobacco mosaic virus genus), pomegranate mosaic virus (PepMV, potato X virus genus), tomato spotted wilt virus (TSWV, tomato spotted wilt virus genus) and tomato ringspot virus (ToRSV, codonopsis mosaic virus family) six pepper-related RNA viruses as detection targets, and combined LAMP with microfluidic chip technology, so that only a single sample addition is required to achieve efficient and accurate detection of multiple pepper viruses at the same time. The advantages of the present invention are simple operation, low equipment requirements, and suitable for rapid and highly sensitive virus detection in a field environment.

[0009] The objective of the present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides a primer composition for simultaneously detecting multiple pepper viruses by loop-mediated isothermal amplification, comprising the following primer sets: a primer set for detecting pepper mild mottle virus (PMMoV), a primer set for detecting tomato brown rugose fruit virus (ToBRFV), a primer set for detecting tomato mottle mosaic virus (ToMMV), a primer set for detecting pomegranate mosaic virus (PepMV), a primer set for detecting tomato spotted wilt virus (TSWV) and a primer set for detecting tomato ringspot virus (ToRSV).

[0011] According to some preferred embodiments of the present invention, the primer composition for simultaneously detecting multiple pepper viruses by loop-mediated isothermal amplification also includes a primer set for detecting pepper rbcL gene.

[0012] According to some embodiments of the present invention, the primer set for detecting PMMoV comprises:

[0013] The outer primer pair PMMoV-F3 and PMMoV-B3 (PMMoV-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 1 and 2, respectively;

[0014] The inner primer pair PMMoV-FIP and PMMoV-BIP (PMMoV-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 3 and 4, respectively; and

[0015] The nucleotide sequences of the loop primer pair PMMoV-LF and PMMoV-LB (PMMoV-LF / LB) are shown in SEQ ID NOs. 5 and 6, respectively.

[0016] According to some embodiments of the present invention, the molar ratio of the outer primer pair PMMoV-F3 / B3, the inner primer pair PMMoV-FIP / BIP and the loop primer pair PMMoV-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0017] According to some embodiments of the present invention, the primer set for detecting ToBRFV comprises:

[0018] The outer primer pair ToBRFV-F3 and ToBRFV-B3 (ToBRFV-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 7 and 8, respectively;

[0019] An inner primer pair ToBRFV-FIP and ToBRFV-BIP (ToBRFV-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 9 and 10, respectively; and

[0020] The nucleotide sequences of the loop primer pair ToBRFV-LF and ToBRFV-LB (ToBRFV-LF / LB) are shown in SEQ ID NOs. 11 and 12, respectively.

[0021] According to some embodiments of the present invention, the molar ratio of the outer primer pair ToBRFV-F3 / B3, the inner primer pair ToBRFV-FIP / BIP and the loop primer pair ToBRFV-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0022] According to some embodiments of the present invention, the primer set for detecting ToMMV comprises:

[0023] The outer primer pair ToMMV-F3 and ToMMV-B3 (ToMMV-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 13 and 14, respectively;

[0024] An inner primer pair ToMMV-FIP and ToMMV-BIP (ToMMV-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 15 and 16, respectively; and

[0025] The nucleotide sequences of the loop primer pair ToMMV-LF and ToMMV-LB (ToMMV-LF / LB) are shown in SEQ ID NOs. 17 and 18, respectively.

[0026] According to some embodiments of the present invention, the molar ratio of the outer primer pair ToMMV-F3 / B3, the inner primer pair ToMMV-FIP / BIP and the loop primer pair ToMMV-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0027] According to some embodiments of the present invention, the primer set for detecting PepMV comprises:

[0028] The outer primer pair PepMV-F3 and PepMV-B3 (PepMV-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 19 and 20, respectively;

[0029] The inner primer pair PepMV-FIP and PepMV-BIP (PepMV-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 21 and 22, respectively; and

[0030] The nucleotide sequences of the loop primer pair PepMV-LF and PepMV-LB (PepMV-LF / LB) are shown in SEQ ID NOs. 23 and 24, respectively.

[0031] According to some embodiments of the present invention, the molar ratio of the outer primer pair PepMV-F3 / B3, the inner primer pair PepMV-FIP / BIP and the loop primer pair PepMV-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0032] According to some embodiments of the present invention, the primer set for detecting TSWV comprises:

[0033] The outer primer pair TSWV-F3 and TSWV-B3 (TSWV-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 25 and 26, respectively;

[0034] an inner primer pair TSWV-FIP and TSWV-BIP (TSWV-FIP / BIP), the nucleotide sequences of which are shown in SEQ ID NOs. 27 and 28, respectively; and

[0035] The nucleotide sequences of the loop primer pair TSWV-LF and TSWV-LB (TSWV-LF / LB) are shown in SEQ ID NOs. 29 and 30, respectively.

[0036] According to some embodiments of the present invention, the molar ratio of the outer primer pair TSWV-F3 / B3, the inner primer pair TSWV-FIP / BIP and the loop primer pair TSWV-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0037] According to some embodiments of the present invention, the primer set for detecting ToRSV comprises:

[0038] The outer primer pair ToRSV-F3 and ToRSV-B3 (ToRSV-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 31 and 32, respectively;

[0039] An inner primer pair ToRSV-FIP and ToRSV-BIP (ToRSV-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 33 and 34, respectively; and

[0040] The nucleotide sequences of the loop primer pair ToRSV-LF and ToRSV-LB (ToRSV-LF / LB) are shown in SEQ ID NOs. 35 and 36, respectively.

[0041] According to some embodiments of the present invention, the molar ratio of the outer primer pair ToRSV-F3 / B3, the inner primer pair ToRSV-FIP / BIP and the loop primer pair ToRSV-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0042] According to some embodiments of the present invention, the primer set for detecting the rbcL gene comprises:

[0043] The outer primer pair rbcL-F3 and rbcL-B3 (rbcL-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 37 and 38, respectively;

[0044] The inner primer pair rbcL-FIP and rbcL-BIP (rbcL-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 39 and 40, respectively; and

[0045] The nucleotide sequences of the loop primer pair rbcL-LF and rbcL-LB (rbcL-LF / LB) are shown in SEQ ID NOs. 41 and 42, respectively.

[0046] According to some embodiments of the present invention, the molar ratio of the outer primer pair rbcL-F3 / B3, the inner primer pair rbcL-FIP / BIP and the loop primer pair rbcL-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0047] In the present invention, each target primer set includes a pair of outer primers, a pair of inner primers and a pair of loop primers, and the molar ratio between the primers in each pair of primers is 1: 1. For example, the molar ratio of PMMoV-F3 and PMMoV-B3 in the outer primer pair PMMoV-F3 / B3 of the PMMoV target is 1: 1, the molar ratio of PMMoV-FIP and PMMoV-BIP in the inner primer pair PMMoV-FIP / BIP is 1: 1, and the molar ratio of PMMoV-LF and PMMoV-LB in the loop primer pair PMMoV-LF / LB is 1: 1.

[0048] In a second aspect, the present invention provides a microfluidic chip for simultaneously detecting multiple pepper viruses, wherein each primer set in the primer composition according to the first aspect of the present invention is respectively contained in different reaction pools of the microfluidic chip.

[0049] According to some embodiments of the present invention, the microfluidic chip further comprises a reaction pool for containing a negative control and / or a positive control.

[0050] According to some preferred embodiments of the present invention, in each group of reaction pools, each primer group in the primer composition according to the first aspect of the present invention is respectively contained in different reaction pools of the microfluidic chip. The microfluidic chip is provided with multiple groups of reaction pools to achieve simultaneous detection of multiple samples, which is fast, simple, synchronous and efficient.

[0051] According to some embodiments of the present invention, each of the reaction pools contains a lyophilized mixture of each primer set, an RNA loop-mediated isothermal amplification premix and a fluorescent dye, wherein the RNA loop-mediated isothermal amplification premix contains reverse transcriptase, DNA polymerase, deoxyribonucleoside triphosphates (dNTPs) and a reaction buffer.

[0052] According to some embodiments of the present invention, each of the reaction pools further comprises a Tte UvrD helicase and an ultra-thermostable single-stranded binding protein (ET SSB); preferably, the Tte UvrD helicase is a Tte UvrD helicase that tolerates high temperatures (70°C); preferably, the ultra-thermostable single-stranded binding protein is an ultra-thermostable single-stranded binding protein that tolerates extremely high temperatures (95°C). The Tte UvrD helicase used in the present invention (e.g., a Tte UvrD helicase that tolerates high temperatures (70°C)) can efficiently open the DNA double-stranded structure during the amplification reaction and improve the reaction specificity; the ultra-thermostable single-stranded binding protein (ET SSB) used in the present invention (e.g., an ultra-thermostable single-stranded binding protein that tolerates extremely high temperatures (95°C)) can stabilize the single-stranded structure, facilitate rapid primer binding, and improve the reaction sensitivity.

[0053] The present invention introduces Tte UvrD helicase and ET SSB which are not used in conventional LAMP technology, and can achieve higher specificity and sensitivity compared with conventional LAMP technology. Among them, the basic principle of LAMP technology integrating Tte UvrD helicase and ET SSB is as follows Figure 2 shown.

[0054] According to some embodiments of the present invention, the microfluidic chip further comprises a vent hole, a sample addition pool, a quantitative pool and a waste liquid pool.

[0055] In the microfluidic chip of the present invention, the air holes are used for air inlet and outlet, and at the same time ensure the subsequent smooth sample loading; the sample loading pool is used for liquid sampling, and the sample loading liquid is a mixture of reaction liquid (such as LAMP reaction liquid) and sample; the quantitative pool is used for liquid quantification to ensure that the reaction volume is fixed; the reaction pool is used for reaction, and a freeze-dried mixture (including a mixture freeze-dried powder of each primer set, RNA loop-mediated isothermal amplification premix and fluorescent dye) is pre-buried in the pool; the waste liquid pool is used to store excess waste liquid.

[0056] According to some preferred embodiments of the present invention, the microfluidic chip further comprises a bottom film bonded to the bottom thereof and a top film bonded to the top thereof, and the bottom film and the top film seal the microfluidic chip.

[0057] According to some preferred embodiments of the present invention, the top film further comprises a sealing film.

[0058] In a third aspect, the present invention provides a method for preparing a microfluidic chip according to the second aspect of the present invention, comprising:

[0059] (1) dissolving each primer set in the primer composition according to the first aspect of the present invention, the RNA loop-mediated isothermal amplification premix and the fluorescent dye in sterile water to obtain a mixed solution;

[0060] (2) spotting the mixed solution into the reaction wells of the microfluidic chip respectively;

[0061] (3) Covering the bottom of the chip with the base film, heating at 45-65° C., preferably 60° C. and 0.1-1 MPa, preferably 0.5 MPa for 0.5-3 minutes, preferably 2 minutes, so that the base film is bonded to the bottom of the chip;

[0062] (4) After freeze-drying, cover the top of the chip with the top film, and heat at 45-65°C, preferably 60°C and 0.1-1 MPa, preferably 0.5 MPa, for 0.5-3 minutes, preferably 2 minutes, to bond the top film to the top of the chip.

[0063] Preferably, the freeze-drying comprises the following steps:

[0064] (4-1) pre-freezing the chip at 1 to 100 kPa and -60 to -40°C, preferably 100 kPa and -50°C for 30 to 200 minutes, preferably 120 minutes, and then drying at 1 to 100 Pa and -60 to -40°C, preferably 100 Pa and -50°C for 30 to 200 minutes, preferably 120 minutes;

[0065] (4-2) Continue drying under a vacuum degree of 1 to 10 Pa, preferably 1 Pa: heat up to -50 to -30 ° C, preferably -40 ° C, keep for 30 to 200 minutes, preferably 120 minutes, then heat up to -40 to -20 ° C, preferably -30 ° C, keep for 30 to 200 minutes, preferably 120 minutes, then heat up to -30 to 0 ° C, preferably -20 ° C, keep for 30 to 200 minutes, preferably 120 minutes, then heat up to 20 to 30 ° C, preferably 25 ° C.

[0066] The microfluidic chip provided by the present invention seals the chip with the bottom film and the top film as a whole through a hot pressing bonding process, wherein the bottom film is used to seal the bottom layer of the chip, and the top film (including the sealing film) is used to seal the top layer of the chip, and the sealing film is used to seal after adding the sample. The hot pressing bonding process includes the film sticking and laminating procedures of the bottom film and the top film, the material used for the bottom film can be plastic, and the material used for the top film (including the sealing film) can be plastic, both of which have good biocompatibility and strong light transmittance, and are suitable for real operations.

[0067] Specifically, the hot pressing bonding process includes attaching a complete circular film 9596R to the bottom of the chip after the spotting is completed, the film pressing temperature is 60°C, the film pressing pressure is 0.5MPa, and the film pressing time is 2min; after the chip is freeze-dried, a circular film 4970c containing a fan-shaped sealing film is attached to the top of the chip, the film pressing temperature is 60°C, the film pressing pressure is 0.5MPa, and the film pressing time is 2min. The freeze-drying process includes placing the chip in a freeze dryer, staying at -50°C for 120min, turning on the vacuum pump (100kPa), staying at -50°C for 120min (vacuum degree 100Pa); turning on the heating, maintaining the vacuum degree at 1Pa, heating to -40°C, maintaining for 120min, continuing to heat to -30°C, maintaining for 120min, then heating to -20°C, maintaining for 120min, heating to 25°C, and closing the freeze dryer. Among them, the bottom film material is plastic, and the top film (including the sealing film) material is plastic. After the chip is freeze-dried, vacuum the aluminum bag to package the chip, and then place it in a 25°C constant temperature box overnight.

[0068] The inventors of the present invention have found through a large number of experiments and studies that other bonding processes have disadvantages to varying degrees: (1) Laser welding has requirements for the properties of the fused material to absorb laser light, and generally requires that the two bonding materials have similar light absorption properties to a certain extent; (2) Solvent bonding has the problem that the glue easily penetrates into the channel and causes the channel to be blocked, and this process has high requirements for the accuracy of the heating temperature and time. Some gas products may also be generated during the curing process, causing pollution to the chip cavity environment; (3) Double-sided adhesive bonding has the disadvantage that the glue penetrates into the channel and causes the channel to be blocked, and double-sided adhesive bonding also has obvious disadvantages such as non-specific adsorption of samples or markers; (4) Single-sided adhesive film bonding has very high requirements for the product quality of the adhesive film, and the bonding pressure is difficult to control. The commonly used film bonding process is manual film scraping, which has a great deal of uncontrollability. For places that cannot be bonded, there is a risk of leakage during the PCR amplification process; (5) Ultrasonic welding has high requirements for the welding line. The uneven welding line will lead to a cold welding point, which is prone to leakage, and the cold welding point cannot be detected.

[0069] The present invention overcomes the above-mentioned shortcomings by adopting the hot pressing bonding method, and also has the following great advantages: first, the microfluidic chip and the PC membrane are bonded at the molecular level mainly by the coordination of bonding temperature, bonding pressure and bonding time, without the need to use any auxiliary adhesives, and there is no bonding blind spot, and no pollutants penetrate and adhere to the microchannel; second, the mechanical properties and thermal properties of the chip treated by hot pressing bonding remain unchanged; third, the cost of the membrane used for hot pressing is relatively low, and the machine design and manufacturing are relatively simple, which is convenient for actual operation.

[0070] In addition, the present invention adopts a pre-freezing and step-by-step temperature freeze-drying process, wherein pre-freezing solidifies free water, giving the product the same shape before and after drying; annealing reshapes the shape and size of ice crystals, prompting subsequent rapid sublimation of water; primary drying (sublimation drying) allows the solidified free water to sublime and escape, and if secondary drying is entered too early, it is easy to cause the product to collapse or melt back; secondary drying (desorption drying) removes part of the bound water, reducing the moisture content to a lower level.

[0071] In a fourth aspect, the present invention provides a kit for simultaneously detecting multiple pepper viruses, comprising the microfluidic chip according to the second aspect of the present invention.

[0072] According to some embodiments of the present invention, the kit further comprises other reagents for loop-mediated isothermal amplification; preferably, the other reagents for loop-mediated isothermal amplification comprise Mg 2+ and enzyme-free water; More preferably, the Mg in the other reagents for loop-mediated isothermal amplification 2+ The concentration is 3~6mM.

[0073] According to some embodiments of the present invention, the kit further comprises a positive control, wherein the positive control comprises the following pepper virus targets and target amplified gene fragments of pepper rbcL gene: PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV; preferably, the nucleotide sequences of the PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV targets and target amplified gene fragments of pepper rbcL gene are shown in SEQ ID NOs. 115 to 121, respectively. The freeze-dried plasmid powder constructed using PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV targets and target amplified gene fragments of pepper rbcL gene is placed in the kit.

[0074] The nucleotide sequences of the target amplified gene fragments of PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV targets and the pepper rbcL gene of the present invention are shown in the following table:

[0075]

[0076] According to some embodiments of the invention, the kit further comprises a scraper.

[0077] The detection kit of the present invention, which is based on LAMP and combined with microfluidic technology, can achieve efficient and accurate detection of multiple pepper viruses at the same time with only a single sample addition. It can also be superimposed with the addition of Tte UvrD helicase that tolerates high temperatures (70°C) and ultra-thermostable single-stranded binding protein (ET SSB) that tolerates extremely high temperatures (95°C), thereby having higher specificity and sensitivity than using conventional LAMP technology.

[0078] In a fifth aspect, the present invention provides a method for simultaneously detecting multiple pepper viruses, which comprises using the microfluidic chip according to the second aspect of the present invention or the kit according to the fourth aspect of the present invention for detection.

[0079] According to some embodiments of the present invention, the method includes performing a loop-mediated isothermal amplification reaction on the sample to be tested using the microfluidic chip according to the second aspect of the present invention or the kit according to the fourth aspect of the present invention, and determining the viral infection status of the sample to be tested based on the generated fluorescence curve.

[0080] According to some embodiments of the present invention, the temperature of the loop-mediated isothermal amplification reaction is 60°C to 65°C, preferably 62°C.

[0081] According to some embodiments of the present invention, the loop-mediated isothermal amplification reaction time is 30 to 60 minutes, preferably 45 minutes.

[0082] According to some preferred embodiments of the present invention, the target sequence concentration of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV or rbcL gene in the sample to be tested is not less than 5000 copies / mL, more preferably not less than 1000 copies / mL. In other words, the detection method of the present invention using the above-mentioned microfluidic chip or kit has a minimum detection limit of 5000 copies / mL, preferably 1000 copies / mL, for the various pepper virus RNAs.

[0083] In the present invention, the pepper virus infection is judged based on the target sequence amplification results of the pepper endogenous gene rbcL and the PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV genes. When the gene rbcL in the fluorescence curve detected shows an S-shaped curve and any one or more of the PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV virus detection targets show an S-shaped curve, it means that the sample to be tested is a pepper sample and there is one or more corresponding pepper-related virus infection; when the PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV virus detection indicators do not show an S-shaped curve, it means that these six types of pepper-related viruses are not present in the sample to be tested.

[0084] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0085] 1. The present invention is based on LAMP and combined with microfluidics technology, and a single sample addition can achieve simultaneous detection of up to six related viruses of pepper;

[0086] 2. The microfluidic chip of the present invention has the advantages of simple operation, uniform liquid separation, good biocompatibility, and environmental friendliness. After being further designed with multiple regions, it can detect multiple samples at the same time, and can also have the function of simultaneously detecting multiple targets by adding samples to a single region;

[0087] 3. The detection kit of the present invention can achieve amplification quickly and efficiently, so that the entire isothermal amplification reaction can be completed within 45 minutes, and the detection result can be directly judged by detecting the fluorescent signal and the peak time of the fluorescent signal, and the result judgment is simple. The recognition of the 6 specific regions of the corresponding virus detection target sequence by the 4 inner and outer primers corresponding to each detection target and the improvement of the amplification efficiency by the 2 loop primers ensure the high specificity of the chip isothermal amplification, with strong specificity, and its minimum detection limit is 1000 copies / mL, with high sensitivity;

[0088] 4. The detection kit of the present invention further uses Tte UvrD helicase that can withstand high temperature (70°C) and ultra-thermostable single-stranded binding protein (ET SSB) that can withstand extremely high temperature (95°C), which has higher specificity and sensitivity than conventional LAMP technology.

[0089] When the microfluidic chip or detection kit of the present invention is used for pepper virus detection, it can detect pepper-related viruses and determine the types of virus infection under isothermal conditions quickly, simply, synchronously, efficiently, highly specifically and sensitively, providing a new technical platform for the detection of pepper-related viruses and the determination of infection types, and providing a fast and highly accurate detection means for field epidemic monitoring and port quarantine of Solanaceae agricultural products. The microfluidic chip or detection kit of the present invention can also be used for grassroots agricultural product testing units and various import and export quarantine inspection and prevention control centers to screen and detect pepper-related viruses, and provide guidance for the selection of subsequent detoxification treatment or other treatment methods, with broad market prospects and greater economic and social benefits, and suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0091] Figure 1 A schematic diagram of the structure of the microfluidic chip provided in Example 1 of the present invention;

[0092] Figure 2A basic schematic diagram showing the LAMP technology of the present invention integrating Tte UvrD helicase and ET SSB;

[0093] Figure 3 For Example 3 of the present invention, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of PMMoV primer set were screened using PMMoV plasmid solution with a copy / mL concentration as a template;

[0094] Figure 4 For Example 3 of the present invention, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the ToBRFV primer set were screened using a ToBRFV plasmid solution with a copy / mL concentration as a template;

[0095] Figure 5 For Example 3 of the present invention, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the ToMMV primer set were screened using a ToMMV plasmid solution with a copy / mL concentration as a template;

[0096] Figure 6 For Example 3 of the present invention, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the PepMV primer set were screened using a PepMV plasmid solution with a copy / mL concentration as a template;

[0097] Figure 7 For Example 3 of the present invention, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of TSWV primer sets were screened using TSWV plasmid solution with a copy / mL concentration as a template;

[0098] Figure 8 For Example 3 of the present invention, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the ToRSV primer set were screened using a ToRSV plasmid solution with a copy / mL concentration as a template;

[0099] Fig. 9 For Example 3 of the present invention, negative control (NC) and 10 4 , 10 5and 10 6 The isothermal amplification test results of the rbcL primer set were screened using a rbcL plasmid solution with a copy / mL concentration as a template;

[0100] Fig.10 To show the target-specific isothermal amplification detection results of Example 4 of the present invention using the mixed nucleic acid of genomic RNA of CMV, TMV and ToMV as a template to detect PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV;

[0101] Fig.11 To show the target-specific isothermal amplification detection results of Example 4 of the present invention for detecting PMMoV using the genomic RNA of six related viruses of pepper as templates;

[0102] Fig.12 To show the target-specific isothermal amplification detection results of Example 4 of the present invention using the genomic RNA of six related viruses of pepper as templates to detect ToBRFV;

[0103] Fig.13 To show the target-specific isothermal amplification detection results of Example 4 of the present invention using the genomic RNA of six related viruses of pepper as templates to detect ToMMV;

[0104] Fig.14 To show the target-specific isothermal amplification detection results of Example 4 of the present invention using the genomic RNA of six related viruses of pepper as templates to detect PepMV;

[0105] Fig.15 To show the target-specific isothermal amplification detection results of Example 4 of the present invention for detecting TSWV using the genomic RNA of six related viruses of pepper as templates;

[0106] Fig.16 To show the target-specific isothermal amplification detection results of Example 4 of the present invention using the genomic RNA of six related viruses of pepper as templates to detect ToRSV;

[0107] Fig.17 To show the target-specific isothermal amplification detection results of Example 4 of the present invention for detecting the rbcL gene using the whole genome RNA of pepper, tomato, soybean, corn, rice, barley, wheat seeds and potato tubers as templates;

[0108] Fig.18 To show that Example 5 of the present invention uses a detection kit 1 ( Fig.18 A) and detection kit II ( Fig.18B) Isothermal amplification assay results for target sensitivity of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV, and rbcL at a mixed plasmid concentration of 5000 copies / mL.

[0109] Fig.19 To show that Example 5 of the present invention uses a detection kit 1 ( Fig.19 A) and detection kit II ( Fig.19 B) Isothermal amplification assay results showing target sensitivity for PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV, and rbcL at a mixed plasmid concentration of 1000 copies / mL.

[0110] Fig. 20 To show that Example 5 of the present invention uses a detection kit 1 ( Fig. 20 A) and detection kit II ( Fig. 20 B) Isothermal amplification assay results of target sensitivity for PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV, and rbcL at a mixed plasmid concentration of 500 copies / mL;

[0111] Fig.21 The isothermal amplification detection results of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL targets of Xiangyan sample ① detected in Example 6 of the present invention;

[0112] Fig. 22 The isothermal amplification detection results of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL targets of Xiangyan sample ② detected in Example 6 of the present invention;

[0113] Fig.23 The isothermal amplification detection results of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL targets of Shanxi Academy of Agricultural Sciences sample ③ detected in Example 6 of the present invention;

[0114] Fig.24 This is the isothermal amplification detection result of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL targets of Shanxi Academy of Agricultural Sciences sample ④ detected in Example 6 of the present invention. DETAILED DESCRIPTION

[0115] The present invention is described in further detail below in conjunction with specific embodiments, and the present invention can be better understood according to the following examples. However, it should be readily understood by those skilled in the art that the following examples are merely illustrative and are not intended to limit the present invention to these specific embodiments. It should be appreciated by those skilled in the art that the present invention will encompass all improvements, alternatives and equivalents included within the scope of the claims.

[0116] Approximate terms in the specification and claims of this application are used to modify quantities, which means that the present invention is not limited to the specific quantity, but also includes the modified parts that are close to the quantity and acceptable but do not cause the relevant basic functions to change. In the specification and claims of this application, range limits can be combined and / or interchanged. If not otherwise specified, these ranges also include all values ​​and sub-ranges contained therein.

[0117] In the following examples, the plasmids used were synthesized by GenScript Biotech Co., Ltd.; the non-target virus CMV, TMV, ToMV genomic RNA and the six related target viruses PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV genomic RNA of pepper were provided free of charge by Changsha Customs of the People's Republic of China (hereinafter referred to as Changsha Customs); the pepper, tomato, soybean, corn, rice, barley, wheat seeds and potato tuber whole genome RNA used were obtained by extracting nucleic acid from commercial grains using a plant tissue virus genome RNA extraction kit; the pepper seed samples ① and ② used were provided free of charge by Hunan Xiangyan Seed Co., Ltd. (hereinafter referred to as Xiangyan); the pepper seed samples ③ and ④ used were provided free of charge by Shanxi Academy of Agricultural Sciences (hereinafter referred to as Shanxi Academy of Agricultural Sciences); the plant tissue virus genome RNA extraction kit used was a commercial kit (DP452) of Tiangen Biotechnology Co., Ltd.; the microfluidic chip supporting instrument used was a microfluidic nucleic acid analyzer BHP9609 produced by Hunan Rongjian Biotechnology Co., Ltd.

[0118] Example 1 Microfluidic Chip

[0119] The microfluidic chip provided in this embodiment includes a chip and a bottom film and a top film (including a sealing film) bonded together with the chip. Figure 1 The schematic diagram of the structure of the microfluidic chip provided in this embodiment is shown. The chip body is divided into four areas A / B / C / D, each of which is composed of a vent hole 1, a sample loading pool 2, a quantitative pool 3, a reaction pool 4 and a waste liquid pool 5. The vent hole 1 is used for air inlet and outlet, and at the same time ensures smooth subsequent sample loading; the sample loading pool 2 is used for liquid loading, and the loading liquid is a mixture of isothermal amplification reaction liquid and sample; the quantitative pool 3 is used for liquid quantification to ensure that the reaction volume is fixed; the reaction pool 4 is used for reaction, and the mixture freeze-dried powder is pre-buried in the pool; the waste liquid pool 5 is used to store excess waste liquid.

[0120] In this embodiment, two different microfluidic chips are prepared. The difference between the two microfluidic chips is that the mixture freeze-dried powder pre-embedded in the reaction pool 4 is different. The pre-embedded mixture freeze-dried powder specifically includes the following two methods:

[0121] Method 1 includes: ① a primer set designed according to the specific conserved gene sequence of the target gene to be detected (the detection targets are: PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL), ② a commercially available lyophilizable RNALAMP Mix premix (a lyophilizable reaction solution containing reverse transcriptase, Bst DNA polymerase, reaction buffer, dNTPs and other auxiliary materials) and ③ a fluorescent dye (SYBR Green I).

[0122] Method 2 includes: ① a primer set designed according to the specific conserved gene sequence of the target gene to be detected (the detection targets are: PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL), ②Tte UvrD helicase, ③ETSSB, ④ a commercially available lyophilizable RNA LAMP Mix premix (a lyophilizable reaction solution containing reverse transcriptase, Bst DNA polymerase, reaction buffer, dNTPs and other auxiliary materials) and ⑤ a fluorescent dye (SYBR Green I).

[0123] The Tte UvrD helicase used in this example is the M1202 reagent produced by NEB, the ET SSB is the M2401 reagent produced by NEB, and the commercial lyophilizable RNA LAMP Mix premix is ​​the MDX118 reagent produced by Meridian, with a final concentration of 1X.

[0124] Among the various pre-embedded primer sets, the molar ratio of PMMoV-F3 / B3:PMMoV-FIP / BIP:PMMoV-LF / LB was 1:8:4; the molar ratio of ToBRFV-F3 / B3:ToBRFV-FIP / BIP:ToBRFV-LF / LB was 1:8:4; the molar ratio of ToMMV-F3 / B3:ToMMV-FIP / BIP:ToMMV-LF / LB was 1:8:4; and the molar ratio of PepMV-F3 / B3:Pe The molar ratio of pMV-FIP / BIP: PepMV-LF / LB is 1:8:4; the molar ratio of TSWV-F3 / B3: TSWV-FIP / BIP: TSWV-LF / LB is 1:8:4; the molar ratio of ToRSV-F3 / B3: ToRSV-FIP / BIP: ToRSV-LF / LB is 1:8:4; the molar ratio of rbcL-F3 / B3: rbcL-FIP / BIP: rbcL-LF / LB is 1:8:4. Each target primer set includes a pair of outer primers, a pair of inner primers and a pair of loop primers, and the molar ratio between primers in each pair of primers in the same group is 1:1.

[0125] The primer set corresponding to the detection target, the commercially available lyophilizable RNA LAMP Mix premix and the fluorescent dye (the microfluidic chip of method 2 is based on the microfluidic chip of method 1 and Tte UvrD helicase and ET SSB are added) are dissolved in sterile water to obtain a lyophilized mixture. Among them, the molar ratio between the primer pairs in each primer set is as shown above, the final concentration of Tte UvrD helicase in the microfluidic chip of method 2 is 0.1ng / μL, the final concentration of ET SSB is 5ng / μL, the final concentration of the commercially available lyophilizable RNA LAMP Mix premix is ​​1X, and the final concentration of the fluorescent dye is 1X. The corresponding lyophilized mixture is spotted into the corresponding target well of reaction pool 4 for bonding and lyophilization.

[0126] Bonding process: After the sample is spotted, a complete circular film 9596R is attached to the bottom, the film pressing temperature is 60℃, the film pressing pressure is 0.5MPa, and the film pressing time is 2min; after the chip is freeze-dried, a circular film 4970c with a fan-shaped sealing film is attached to the top, the film pressing temperature is 60℃, the film pressing pressure is 0.5MPa, and the film pressing time is 2min. Freeze-drying process: After the chip is placed in the freeze dryer, stay at -50℃ for 120min, turn on the vacuum pump (100kPa), stay at -50℃ for 120min (vacuum degree 100Pa); turn on the heating, maintain the vacuum degree 1Pa, heat up to -40℃, maintain for 120min, continue to heat up to -30℃, maintain for 120min, then heat up to -20℃, maintain for 120min, heat up to 25℃, and turn off the freeze dryer. Among them, the bottom film material is plastic, and the top film (including the sealing film) material is plastic. After the chip is freeze-dried, vacuum the aluminum bag to package the chip, and then place it in a 25°C constant temperature box overnight to obtain two types of microfluidic chips.

[0127] Example 2 Detection Kit

[0128] The components and compositions of the two detection kits provided in this embodiment are shown in Table 1 below:

[0129] Table 1

[0130]

[0131] Example 3 Primer screening experiment

[0132] Each set of primers is designed according to the specific conserved sequence of the genome of each detection target, wherein each set of primers includes: loop primers LF and / or LB, outer primers B3 and F3, inner primers FIP and BIP. In order to obtain a primer set with optimal specificity and sensitivity, the inventors of the present invention designed a primer set according to the specific conserved gene sequence of the target gene to be detected, and obtained three series of primer compositions as shown in Table 2, Table 3 and Table 4 below:

[0133] Table 2 Screening primer series 1 (the curve icons are left triangle / five-pointed star / asterisk / sphere, and the left triangle curve represents the negative control)

[0134]

[0135] Table 3 Screening primer series 2 (curve icons are right triangle / diamond / pentagon / square, right triangle curve represents negative control)

[0136]

[0137] Table 4 Screening primer sequence 3 (curve icons are cross / lower triangle / hexagon / upper triangle, the cross curve represents negative control)

[0138]

[0139] Primer screening experiment:

[0140] The reaction system was prepared according to Table 5.

[0141] Table 5

[0142]

[0143] Enzyme-free water was used as solvent to prepare 10 samples of six pepper-related viruses: PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV, and the pepper endogenous gene rbcL as the detection target. 10 10 copies / mL concentration of plasmid solution. 10 The plasmid solution with a concentration of 10 copies / mL was diluted to make the concentration of the solution 10 4 copies / mL, 10 5 copies / mL and 10 6 copies / mL.

[0144] The mixed plasmid solution diluted in various gradients was used as the template, and RNase-free water was used as the negative control (NC). The above primer combinations were placed in the detection kit 2 prepared in Example 2, mixed in the isothermal amplification reagent, and the reaction system was prepared according to Table 5 above. The samples were added to the sample pools respectively, the sealing film on the top film was torn off and fitted tightly, and a scraper was used to scrape in the same direction until it was completely fitted, and the chip was placed on the tray of the microfluidic nucleic acid analyzer BHP9609 with the reaction holes facing up and fixed. After centrifugation, the isothermal amplification reaction was carried out at 62°C for 45 minutes. The fluorescence curve detection results are shown in the following figure. Figures 3 to 9 As shown, three series of primer sets provided in Tables 2-4 were screened for 7 detection targets, wherein, for primer sequence 1 in Table 2 above, Figure 3-9 The left triangle curve in the figure represents the negative control, and the five-pointed star curve represents the 10% of the primer sequence 1. 4 Copies / mL concentration, the asterisk curve represents 10 of the primer sequence 1 5Copies / mL concentration, the spherical curve represents 10 of the primer sequence 6 Copies / mL concentration; for primer sequence 2 in Table 3 above, Figure 3-9 The right triangle curve in the figure represents the negative control, and the diamond curve represents the 10 4 The pentagonal curve represents 10 copies / mL of primer sequence 2. 5 The square curve represents 10 copies / mL of primer sequence 2. 6 Copies / mL concentration; for primer sequence three in Table 4 above, Figure 3-9 The cross curve in the middle represents the negative control, and the lower triangle curve represents the 10% of primer sequence three. 4 Copies / mL concentration, the hexagonal curve represents 10 of primer sequence three 5 copies / mL concentration, the upper triangle curve represents 10 of primer sequence three 6 The results are analyzed as follows:

[0145] Figure 3 For Example 3, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of PMMoV primer sets were screened using PMMoV plasmid solution with a concentration of 10 copies / mL as template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions, and the primer series three had only 10 6 The detection time of primer series 2 was later than that of primer series 1 at each concentration, indicating that primer series 1 had the highest sensitivity in detecting PMMoV target and would be used as the preferred primer set in the future.

[0146] Figure 4 For Example 3, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the ToBRFV primer set were screened using a ToBRFV plasmid solution with a concentration of 10 copies / mL as a template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions, and the primer series three was only at 10 6 The copy / mL concentration was detected later, and the primer series 2 was detected at 10 4 The copies / mL concentration could not be detected, but primer series one could be detected at all concentrations, and the detection time was very early, indicating that primer series one had the highest sensitivity in detecting ToBRFV targets and would be used as the preferred primer set in the future.

[0147] Figure 5 For Example 3, negative control (NC) and 10 4 , 105 and 10 6 The isothermal amplification test results of the ToMMV primer set were screened using a ToMMV plasmid solution with a concentration of 10 copies / mL as a template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. The primer series three was 4 The copies / mL concentration could not be detected, and the primer series 2 was detected later than the primer series 1 at each concentration, indicating that the primer series 1 had the highest sensitivity in detecting the ToMMV target and was subsequently used as the preferred primer set.

[0148] Figure 6 For Example 3, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the PepMV primer set were screened using a PepMV plasmid solution with a concentration of 10 copies / mL as a template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. The primer series three was positive at 10 4 The copy / mL concentration was not detected, and the primer series 2 was only 10 6 Primer series 1 could detect PepMV at all concentrations and the detection time was very early, indicating that primer series 1 had the highest sensitivity in detecting PepMV targets and would be used as the preferred primer set in the future.

[0149] Figure 7 For Example 3, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of TSWV primer sets were screened using TSWV plasmid solutions with a concentration of 10 copies / mL as templates. The fluorescence curves showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions, and primer series 2 only had a negative reaction at 10 6 The detection time of primer series three was later than that of primer series one at each concentration, indicating that primer series one had the highest sensitivity in detecting TSWV target and would be used as the preferred primer set in the future.

[0150] Figure 8 For Example 3, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the ToRSV primer set were screened using a ToRSV plasmid solution with a concentration of 10 copies / mL as a template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions, and the primer series three was only at 10 6 Copies / mL concentration detection, primer series 2 at 10 4The copies / mL concentration could not be detected, but primer series one could be detected at all concentrations, and the detection time was very early, indicating that primer series one had the highest sensitivity in detecting ToRSV targets and would be used as the preferred primer set in the future.

[0151] Fig. 9 For Example 3, negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the rbcL primer set were screened using the rbcL plasmid solution with a concentration of 10 copies / mL as the template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. The primer series three and primer series two were positive at 10 4 The primer series 1 could detect the target rbcL at all concentrations, and the detection time was very early, indicating that the primer series 1 had the highest sensitivity in detecting the rbcL target and would be used as the preferred primer set in the future.

[0152] Example 4 Detection Kit Specificity Detection

[0153] The genomic RNA of non-target viruses CMV (cucumber mosaic virus), TMV (tobacco mosaic virus), and ToMV (tomato mosaic virus) were used as templates, or the genomic RNA of six related target viruses of pepper: PMMoV, ToBRFV, ToMMV, PepMV, TSWV, and ToRSV were used as templates, or the whole genome RNA of pepper, tomato, soybean, corn, rice, barley, wheat seeds and potato tubers were used as templates (the genomic RNA of the corresponding virus detection target was used as the positive control, and the genomic RNA of the other five virus detection targets was used as the non-specific target nucleic acid; the whole genome RNA of pepper seeds was used as the positive control, and the whole genome RNA of tomato, soybean, corn, rice, barley, wheat seeds and potato tubers was used as the non-specific target nucleic acid).

[0154] Prepare the reaction system according to Table 5 above, use the nucleic acid detection kit 2 provided in Example 2 for isothermal amplification detection, respectively place the above template RNA in the isothermal amplification reagent provided in the kit and mix, add the sample to the sample pool, tear off the sealing film on the top film to fit tightly, use a scraper to scrape in the same direction until it fits completely, place the chip reaction well upward and stably on the tray of the microfluidic nucleic acid analyzer BHP9609, fix it, centrifuge it, and perform isothermal amplification reaction at 62°C for 45 minutes. The fluorescence curve detection results are shown in the figure. Figures 10 to 17 The result analysis is shown below:

[0155] Fig.10To show the target-specific isothermal amplification detection results of PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV using the genomic RNA of CMV, TMV and ToMV as a mixed nucleic acid template in Example 4. The fluorescence curve shows that none of the detection indicators peaked, indicating that no isothermal amplification reaction occurred in all the detection targets, indicating that the isothermal amplification detection method of the present invention for detecting the targets PMMoV, ToBRFV, ToMMV, PepMV, TSWV and ToRSV has high specificity and does not react with other non-target viruses CMV, TMV and ToMV genomic RNA.

[0156] Fig.11 This is to show the target-specific isothermal amplification detection results of PMMoV using the genomic RNA of six related viruses of pepper as templates in Example 4. The fluorescence curve shows that only the PMMoV curve presents a standard S-type, and no peaks appear in other detection target templates, indicating that only the PMMoV target undergoes an isothermal amplification reaction, indicating that the isothermal amplification detection method of the present invention has a high specificity for the detection target PMMoV and can specifically detect the PMMoV target.

[0157] Fig.12 To show the target-specific isothermal amplification detection results of ToBRFV using the genomic RNA of six related viruses of pepper as templates in Example 4. The fluorescence curve shows that only the ToBRFV curve is a standard S-type, and no peaks appear in other detection target templates, indicating that only the ToBRFV target has undergone an isothermal amplification reaction. This shows that the isothermal amplification detection method of the present invention has a high specificity for the detection target ToBRFV, and can specifically detect the ToBRFV target.

[0158] Fig.13 To show the target-specific isothermal amplification detection results of ToMMV using the genomic RNA of six related viruses of pepper as templates in Example 4. The fluorescence curve shows that only the ToMMV curve presents a standard S-shape, and no peaks appear in other detection target templates, indicating that only the ToMMV target undergoes an isothermal amplification reaction. This indicates that the isothermal amplification detection method of the present invention has a high specificity for the detection target ToMMV, and can specifically detect the ToMMV target.

[0159] Fig.14The isothermal amplification detection results of Example 4 using the genomic RNA of six related viruses of pepper as templates to detect PepMV target specificity. The fluorescence curve shows that only the PepMV curve presents a standard S-type, and no peaks appear in other detection target templates, indicating that only the PepMV target undergoes an isothermal amplification reaction. This indicates that the isothermal amplification detection method of the present invention has a high specificity for the detection target PepMV and can specifically detect the PepMV target.

[0160] Fig.15 The isothermal amplification detection results of Example 4 using the genomic RNA of six related viruses of pepper as templates to detect TSWV are shown. The fluorescence curve shows that only the TSWV curve presents a standard S-shape, and no peaks appear in other detection target templates, indicating that only the TSWV target undergoes an isothermal amplification reaction. This indicates that the isothermal amplification detection method of the present invention has a high specificity for the detection target TSWV and can specifically detect the TSWV target.

[0161] Fig.16 To show the target-specific isothermal amplification detection results of ToRSV using the genomic RNA of six related viruses of pepper as templates in Example 4. The fluorescence curve shows that only the ToRSV curve is a standard S-type, and no peaks appear in other detection target templates, indicating that only the ToRSV target undergoes an isothermal amplification reaction. This shows that the isothermal amplification detection method of the present invention has a high specificity for the detection target ToRSV, and can specifically detect the ToRSV target.

[0162] Fig.17 To show the isothermal amplification detection results of the target specificity of rbcL detected by using the whole genome RNA of pepper, tomato, soybean, corn, rice, barley, wheat seeds and potato tubers as templates in Example 4. The fluorescence curve shows that only the rbcL curve using the whole genome RNA of pepper seeds as a template is a standard S-type, and the other rbcL curves using the whole genome RNA of non-pepper seeds as templates have no peaks, indicating that only the rbcL target of pepper has undergone an isothermal amplification reaction, and the rbcL target has been detected, and the rbcL target has not been detected in other non-pepper seeds. It shows that the detection target rbcL of the present invention is an endogenous gene of pepper and has high specificity.

[0163] Example 5: Sensitivity test of the detection kit

[0164] Six pepper-related viruses: PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and the pepper endogenous gene rbcL were prepared as 10 10 Take 10 μL of each detection target plasmid solution, add 30 μL of enzyme-free water to 100 μL to obtain 10 copies / mL concentration plasmid solution.9 The mixed plasmid solution was prepared at a concentration of 10 copies / mL. 2 times, 10 3 times, 10 4 times, 10 5 times), and then diluted 10 5 The plasmid solution of 10000 copies / mL was diluted in a gradient of 2, 10, and 20 times, so that the concentration of the mixed plasmid solution of each detection target was 5000 copies / mL, 1000 copies / mL, and 500 copies / mL, respectively.

[0165] The mixed plasmid solutions diluted in various gradients were used as templates, and RNase-free water was used as negative control, and the reaction systems were prepared according to Table 5 above. The nucleic acid detection kit 1 and the detection kit 2 provided in Example 2 were used for isothermal amplification detection, respectively. The above templates were placed in the isothermal amplification reagents provided in the kits and mixed, and the samples were added to the sample pool. The sealing film on the top film was torn off and fitted tightly. A scraper was used to scrape in the same direction until it was completely fitted. The chip was placed on the tray of the microfluidic nucleic acid analyzer BHP9609 with the reaction holes facing up and fixed. After centrifugation, the isothermal amplification reaction was carried out at 62°C for 45 minutes. The fluorescence curve detection results are shown in the figure. Figures 18 to 20 The results are analyzed as follows:

[0166] Fig.18 A and Fig.18 B shows the isothermal amplification test results of the target sensitivity of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL using test kit 1 and test kit 2 at a mixed plasmid concentration of 5000 copies / mL. The fluorescence curves show that the negative controls are all negative curves, and the curves of all other detection targets are standard S-shaped, indicating that all detection targets have undergone isothermal amplification reactions at a mixed plasmid concentration of 5000 copies / mL, and all detection targets can be stably detected, but Fig.18 All target curves in A are compared Fig.18 The peak times of the corresponding target curves in B were delayed, indicating that the detection effect of the kit superimposed with Tte UvrD helicase and ultra-thermostable single-stranded binding protein was better at this plasmid concentration.

[0167] Fig.19 A and Fig.19 B shows the isothermal amplification test results of the target sensitivity of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL using the detection kit 1 and the detection kit 2 at a mixed plasmid concentration of 1000 copies / mL. The fluorescence curves show that the negative controls are all negative curves. Fig.19All the detection target curves in B showed a standard S-shape, indicating that all detection targets underwent isothermal amplification reaction at a mixed plasmid concentration of 1000 copies / mL, and all detection targets could be stably detected; Fig.19 The curves of PepMV and TSWV targets in A are non-S-shaped, while the curves of all other detection targets are standard S-shaped, and the detection time of all detection targets is relatively Fig.19 The corresponding target curve in B was delayed by about 5 minutes, indicating that the detection targets PepMV and TSWV were unstable at this plasmid concentration, and the detection effect of each detection target was deteriorated. Fig.19 B. All detection targets can be stably detected at this plasmid concentration.

[0168] Fig. 20 A and Fig. 20 B shows the isothermal amplification test results of the target sensitivity of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL using the detection kit 1 and the detection kit 2 at a mixed plasmid concentration of 500 copies / mL. The fluorescence curves show that the negative controls are all negative curves. Fig. 20 The fluorescence curve in B shows that TSWV is a negative curve, and the curves of other targets are all non-S-type, indicating that at a mixed plasmid concentration of 500 copies / mL, the TSWV target is not amplified, and the amplification of other detection targets is not obvious. Fig. 20 The fluorescence curve in A shows that ToMMV, PepMV, and TSWV are negative curves, and the curves of other targets are slightly upturned, indicating that at a mixed plasmid concentration of 500 copies / mL, ToMMV, PepMV, and TSWV targets are not amplified, and other detection targets are relatively Fig. 20 The corresponding target amplification in B is even less obvious. This indicates that the closer to the sensitivity detection limit, the more obvious the effect of the superimposed Tte UvrD helicase and ultra-thermostable single-stranded binding protein in the microfluidic chip kit.

[0169] From the above test results, it can be seen that the minimum detection limit of the detection target of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL provided by the detection kit one of the present invention is 5000 copies / mL; the minimum detection limit of the detection target of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL provided by the detection kit two of the present invention is 1000 copies / mL, which has higher sensitivity. Compared with the nucleic acid detection kit one without superimposed high temperature tolerant Tte UvrD helicase and extremely high temperature tolerant ET SSB, the nucleic acid detection kit two superimposed with high temperature tolerant Tte UvrD helicase and extremely high temperature tolerant ET SSB is more stable and has an earlier detection time at concentrations of 5000 copies / mL, 1000 copies / mL and 500 copies / mL.

[0170] Example 6 Detection of pepper seed samples

[0171] A commercial plant tissue RNA rapid extraction kit (Tiangen DP452) was used to extract the total RNA from four random pepper seeds provided by Xiangyan and Shanxi Academy of Agricultural Sciences (numbered Xiangyan sample ①, Xiangyan sample ②, Shanxi Academy of Agricultural Sciences sample ③ and Shanxi Academy of Agricultural Sciences sample ④, respectively). The RNA was mixed with the isothermal amplification reaction solution, and the reaction system was prepared according to the above Table 5. The nucleic acid detection kit 2 provided in Example 2 was used to perform isothermal amplification detection. The total RNA of the four pepper seeds was respectively placed in the isothermal amplification reagent provided in the kit and mixed, and then added to the sample pool. The sealing film on the top film was torn off and fit tightly. It was scraped in the same direction with a scraper until it fits completely. The chip reaction well was placed and fixed on the tray of the microfluidic nucleic acid analyzer BHP9609 with the reaction well facing up. After centrifugation, the isothermal amplification reaction was carried out at 62°C for 45 minutes. The fluorescence curve detection results are shown in the following figure. Figures 21 to 24 The result analysis is shown below:

[0172] Fig.21 The isothermal amplification detection results of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL targets for detecting Xiangyan sample ① in Example 6. The fluorescence curve shows that the negative control is a negative curve, the PMMoV and rbcL detection target curves are both standard S-shaped, and no peaks appear in other targets, indicating that only PMMoV and rbcL detection targets have undergone isothermal amplification reactions, and the pepper rbcL endogenous gene is detected in Xiangyan sample ① and PMMoV virus infection exists.

[0173] Fig. 22The isothermal amplification detection results of PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL targets for detecting Xiangyan sample ② in Example 6. The fluorescence curve shows that the negative control is a negative curve, the PMMoV, TSWV and rbcL detection target curves are all standard S-shaped, and the other targets have no peaks, indicating that only PMMoV, TSWV and rbcL detection targets have undergone isothermal amplification reactions, and the pepper rbcL endogenous gene was detected in Xiangyan sample ② and PMMoV and TSWV virus infection was present.

[0174] Fig.23 The isothermal amplification detection results for detecting PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL targets in Shanxi Academy of Agricultural Sciences sample ③ in Example 6. The fluorescence curve shows that the negative control is a negative curve, the PMMoV and rbcL detection target curves are both standard S-shaped, and no peaks appear in other targets, indicating that only PMMoV and rbcL detection targets have undergone isothermal amplification reactions, and the pepper rbcL endogenous gene was detected in Shanxi Academy of Agricultural Sciences sample ③ and PMMoV virus infection was present.

[0175] Fig.24 The isothermal amplification detection results for detecting PMMoV, ToBRFV, ToMMV, PepMV, TSWV, ToRSV and rbcL targets in Shanxi Academy of Agricultural Sciences sample ④ in Example 6. The fluorescence curve shows that the negative control is a negative curve, the PMMoV and rbcL detection target curves are both standard S-shaped, and no peaks appear in other targets, indicating that only PMMoV and rbcL detection targets have undergone isothermal amplification reactions, and the pepper rbcL endogenous gene was detected in Shanxi Academy of Agricultural Sciences sample ④ and PMMoV virus infection was present.

[0176] It can be seen from the above experimental results that the detection method of the present invention can more accurately determine whether it is infected by these viruses and determine the specific virus type, and can be applied to the rapid, efficient and simple amplification detection of pepper samples. The result interpretation is simple, and it has the advantages of high sensitivity and strong specificity. Compared with the ordinary tubular LAMP, the present invention has the advantages of single sample addition and detection of multiple target genes, and is easy to operate: the template and the reaction reagent only need to be simply mixed to add the sample and seal, and the whole system is easy to operate and controllable; fast and efficient: the whole chip can detect 4 samples at the same time, and the whole isothermal amplification chip detection process can be completed within 45 minutes. Each area only needs a single sample addition to synchronously realize the detection of six viruses, and the specific existence of the virus infection of the pepper sample can be quickly determined; low cost: the basic reagents used are conventional commercial reagents, and the microfluidic chip provides a method for accurately controlling and processing trace samples. The reagent volume required for each reaction pool is as small as 5μL; in addition, it is equipped with an existing technology platform, and the product can be customized according to the actual needs of customers.

[0177] Although various aspects and embodiments of the present invention are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and not for limiting purposes. The scope and spirit of the present invention are determined solely by the appended claims.

Claims

1. A primer combination for simultaneously detecting multiple pepper viruses, characterized in that: The primer composition is used for simultaneously detecting multiple pepper viruses by loop-mediated isothermal amplification and includes the following primer sets: a primer set for detecting pepper mild mottle virus, a primer set for detecting tomato brown wrinkled fruit virus, a primer set for detecting tomato mottle mosaic virus, a primer set for detecting pomegranate mosaic virus, a primer set for detecting tomato spotted wilt virus and a primer set for detecting tomato ringspot virus.

2. The primer composition according to claim 1, characterized in that The primer composition for simultaneously detecting multiple pepper viruses by loop-mediated isothermal amplification also includes a primer set for detecting pepper rbcL gene.

3. The primer composition according to claim 1, characterized in that The primer set for detecting pepper mild mottle virus comprises: The outer primer pair PMMoV-F3 and PMMoV-B3, whose nucleotide sequences are shown in SEQ ID NOs. 1 and 2, respectively; The inner primer pair PMMoV-FIP and PMMoV-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 3 and 4, respectively; and The loop primer pair PMMoV-LF and PMMoV-LB, whose nucleotide sequences are shown in SEQ ID NOs. 5 and 6, respectively; The primer set for detecting tomato brown wrinkled fruit virus comprises: The outer primer pair ToBRFV-F3 and ToBRFV-B3, whose nucleotide sequences are shown in SEQ ID NOs. 7 and 8, respectively; The inner primer pair ToBRFV-FIP and ToBRFV-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 9 and 10, respectively; and The loop primer pair ToBRFV-LF and ToBRFV-LB, whose nucleotide sequences are shown in SEQ ID NOs. 11 and 12, respectively; The primer set for detecting tomato mottle mosaic virus comprises: The outer primer pair ToMMV-F3 and ToMMV-B3, whose nucleotide sequences are shown in SEQ ID NOs. 13 and 14, respectively; The inner primer pair ToMMV-FIP and ToMMV-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 15 and 16, respectively; and Loop primer pair ToMMV-LF and ToMMV-LB, whose nucleotide sequences are shown in SEQ ID NOs. 17 and 18, respectively; The primer set for detecting Pepino Mosaic Virus comprises: The outer primer pair PepMV-F3 and PepMV-B3, whose nucleotide sequences are shown in SEQ ID NOs. 19 and 20, respectively; The inner primer pair PepMV-FIP and PepMV-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 21 and 22, respectively; and The loop primer pair PepMV-LF and PepMV-LB, whose nucleotide sequences are shown in SEQ ID NOs. 23 and 24, respectively; The primer set for detecting tomato spotted wilt virus comprises: The outer primer pair TSWV-F3 and TSWV-B3, whose nucleotide sequences are shown in SEQ ID NOs. 25 and 26, respectively; The inner primer pair TSWV-FIP and TSWV-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 27 and 28, respectively; and The loop primer pair TSWV-LF and TSWV-LB, whose nucleotide sequences are shown in SEQ ID NOs. 29 and 30, respectively; The primer set for detecting tomato ringspot virus comprises: The outer primer pair ToRSV-F3 and ToRSV-B3, whose nucleotide sequences are shown in SEQ ID NOs. 31 and 32, respectively; The inner primer pair ToRSV-FIP and ToRSV-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 33 and 34, respectively; and The nucleotide sequences of the loop primer pair ToRSV-LF and ToRSV-LB are shown in SEQ ID NOs. 35 and 36, respectively.

4. The primer composition according to claim 2, characterized in that The primer set for detecting the pepper rbcL gene comprises: The outer primer pair rbcL-F3 and rbcL-B3, whose nucleotide sequences are shown in SEQ ID NOs. 37 and 38, respectively; The inner primer pair rbcL-FIP and rbcL-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 39 and 40, respectively; and The nucleotide sequences of the loop primer pair rbcL-LF and rbcL-LB are shown in SEQ ID NOs. 41 and 42, respectively.

5. A microfluidic chip for simultaneously detecting multiple pepper viruses, characterized in that: Each primer set in the primer composition according to any one of claims 1 to 4 is respectively contained in different reaction pools of the microfluidic chip.

6. The microfluidic chip according to claim 5, characterized in that: Each primer set in the primer composition according to any one of claims 1 to 4 is respectively contained in different reaction pools of the microfluidic chip.

7. The microfluidic chip according to claim 5, characterized in that: Each of the reaction pools also contains a Tte UvrD helicase and an ultrathermostable single-stranded binding protein.

8. The microfluidic chip according to claim 5, characterized in that: The microfluidic chip also includes a vent hole, a sample addition pool, a quantitative pool and a waste liquid pool.

9. The microfluidic chip according to any one of claims 5 to 8, characterized in that: The microfluidic chip further comprises a bottom film bonded to the bottom thereof and a top film bonded to the top thereof, and the bottom film and the top film seal the microfluidic chip.

10. A method for preparing a microfluidic chip according to any one of claims 5 to 9, characterized in that: The preparation method comprises: (1) dissolving each primer set in the primer composition according to any one of claims 1 to 4, the RNA loop-mediated isothermal amplification premix and the fluorescent dye in sterile water to obtain a mixed solution; (2) spotting the mixed solution into the reaction wells of the microfluidic chip respectively; (3) Cover the bottom of the chip with the base film and heat it at 45~65℃ and 0.1~1Mpa for 0.5~3 minutes to bond the base film to the bottom of the chip; (4) After freeze-drying, cover the top of the chip with the top film and heat it at 45~65℃ and 0.1~1Mpa for 0.5~3 minutes to bond the top film to the top of the chip.

11. The preparation method according to claim 10, characterized in that: The freeze-drying comprises the following steps: (4-1) Pre-freeze the chip at 1-100 kPa and -60--40°C for 30-200 minutes, and then dry it at 1-100 Pa and -60--40°C for 30-200 minutes; (4-2) Continue drying under a vacuum degree of 1 to 10 Pa: heat to -50 to -30°C, maintain for 30 to 200 minutes, then heat to -40 to -20°C, maintain for 30 to 200 minutes, then heat to -30 to 0°C, maintain for 30 to 200 minutes, and then heat to 20 to 30°C.

12. A kit for simultaneously detecting multiple pepper viruses, characterized in that: The kit comprises the microfluidic chip according to any one of claims 5 to 9.

13. The kit according to claim 12, characterized in that The kit also contains positive controls, wherein the positive controls include the following pepper virus targets and target amplified gene fragments of the pepper rbcL gene: pepper mild mottle virus, tomato brown rugose fruit virus, tomato mottle mosaic virus, pomegranate mosaic virus, tomato spotted wilt virus and tomato ringspot virus.

14. The kit according to claim 13, characterized in that The nucleotide sequences of the target amplified gene fragments of the pepper mild mottle virus, tomato brown rugose fruit virus, tomato mottle mosaic virus, pomegranate mosaic virus, tomato spotted wilt virus and tomato ringspot virus targets and the pepper rbcL gene are shown in SEQ ID NOs. 115 to 121, respectively.

15. A method for simultaneously detecting multiple pepper viruses, characterized in that: The method comprises using the microfluidic chip according to any one of claims 5 to 9 or the kit according to any one of claims 12 to 14 for detection.

16. The method according to claim 15, characterized in that The method comprises using the microfluidic chip according to any one of claims 5 to 9 or the kit according to any one of claims 12 to 14 to perform a loop-mediated isothermal amplification reaction on the sample to be tested, and determining the virus infection status of the sample to be tested based on the generated fluorescence curve.

17. The method according to claim 15 or 16, characterized in that The target sequence concentration of pepper mild mottle virus, tomato brown rugose fruit virus, tomato mottle mosaic virus, pomegranate mosaic virus, tomato spotted wilt virus, tomato ringspot virus or rbcL gene in the sample to be tested is not less than 5000 copies / mL.

18. The method according to claim 15 or 16, characterized in that The target sequence concentration of pepper mild mottle virus, tomato brown rugose fruit virus, tomato mottle mosaic virus, pomegranate mosaic virus, tomato spotted wilt virus, tomato ringspot virus or rbcL gene in the sample to be tested is not less than 1000 copies / mL.

Images