TWI326357B - Method and kit for detecting nervous virus infection in fish - Google Patents

Description

1326357 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a virus detection method, and more specifically, to a method for detecting a fish-like infection of a neurovirus. [Prior Art] Fish farming is an important economic industry in the Asia-Pacific region. However, due to the fact that the land is thick and thick, regardless of the land fish or the sea box network, most of them are raised at high density, which often causes serious spread of farmed fish diseases. Among the various fish diseases, the mortality of the fish that invade the nervous system, especially the brain virus, is the most serious. Taking the most important economic fish species in the Asia-Pacific region, grouper, for example, the current viral diseases in grouper culture are caused by nervous necrosis virus and iridescent virus (iridovirus^*, causing a large number of deaths of grouper fry and serious threats. Aquaculture industry. - Y〇shikoshi and Inoue (Yoshikoshi and Inoue, 1990, Journal of Fish Disease 13:69-77) found that most of the dead fish are 6 to 10 mm long fry (juvae M 13 to 25111111 juveniles) The obvious symptoms of diseased fish are deeper body color, loss of balance ability, swirling on the surface of the water, and finally sinking into the bottom of the water, but the young fish with a body length of 4〇mm or more have no serious death or death; by histopathology Observed, it was found that the nerve tissue of the diseased fish produced Pu'er necrosis, and the spinal cord, spinal ganglia and brain cells all had obvious vacuolation and pyknosis, and the cells showed contraction and basophilicity; On the one hand, there are no obvious tissue lesions in the ankle, heart, gastrointestinal tract, liver, pancreas, kidney, spleen, skin and bone muscles. It was found that there were virus particles in the cytoplasm of neurons, nematode cells 117404.doc 1326357 (oligodendrocyte) and astrocyte, and in the nerve fibers, the virus was also found in the cytoplasmic inclusion bodies. It is infected by the polluted water environment and by the poisonous fish production and passed to the next generation. This disease is almost universally spread around the world and is named differently according to different regions. For example, it is called viral nerve in Asia. Viral nervous necrosis, known in Europe as Fish Neuropathy or Fish encephalitis. It is known that there are 36 species of seawater and freshwater fish in the world that suffer from this virus infection. Among the eighteen subjects of the five purposes of scorpion, scorpion, scorpion, scorpion, and blunt, most of them are marine fish (Munday et al., 2002, Journal of Fish Disease 25: 127-142; Chi Et al., 2003, Disease of Aquatic Organism 55:221-228), causing enormous economic losses for dozens of farmed fish species around the world. The methods of infection with necrosis virus are as follows: (a) Observing the brain, notochord or retina of the fish by optical microscopy, but the operation is complicated and difficult to observe; (b) Using electron microscopy to detect whether there are virus particles in the fish. Existence, this method is not only complicated and expensive to operate, but also not suitable for large-scale screening; (Check the serological method to detect viral antigens, such as enzyme-linked immune reaction with recombinant viral sheath protein; this method requires well-trained personnel to operate; (4) The use of molecular biological methods to detect viral nucleic acid, this method usually requires the isolation of viral nucleic acid, also requires well-trained personnel to operate; and (4) cell culture of the virus, the currently available cell strain is applicable to all genotype viruses. And GF_1 which can be applied to the grouper necrosis virus (Chi et al., 1999, 〇) 仙1d 117404.doc 1326357

Disease 22: 173-182), but the virus cultivation operation is complicated and time consuming, and it cannot meet the needs of a large number of screening tests. For the sake of the job, it is necessary for the industry to develop a rapid, easy-to-use and accurate method for detecting fish-borne neuroviruses. SUMMARY OF THE INVENTION One object of the present invention is to provide a method for detecting a fish virus infected with a neurovirus, comprising: (a) homogenizing fish brain tissue in a lysis buffer, wherein the lysis buffer comprises 62.5 mM Tris Η8·3, 95 mM KC1, 3.8 mM MgCU, 12.5 mM dithiothreitol (DTT) and 0.63% detergent; (b) taking the clarified liquid from the homogenized fish brain tissue of step (a); c) adding a primer specific for the neuroviral to the clear liquid of step (b) and performing a reverse transcription polymerase chain reaction; and (d) analyzing the product of the resulting reverse transcription polymerase chain reaction, such as containing A nucleic acid fragment of a neuroviral protein indicates that the fish body is infected with a neurovirus. A further object of the present invention is to provide a kit for detecting a fish body infected with a neurovirus, comprising: a lysis buffer comprising 62.5 mM Tris pH 8.3, 95 mM KC1, 3.8 mMMgCl2, 12.5 mM dithiothreitol and 0.63% detergent; primers specific for the neuroviral; and reagents required for reverse transcription polymerase chain reaction. DETAILED DESCRIPTION OF THE INVENTION 117404.doc -8- 1326357 The present invention utilizes reverse transcription polymerase chain reaction technology to directly homogenize brain and weave, and does not need to isolate virus or viral nucleic acid from infected fish to detect fish body. Whether it is infected with a neurovirus. An object of the present invention is to provide a method for detecting a fish virus infected with a neurovirus, which comprises: (4) homogenizing fish brain tissue in a lysis buffer, #_的溶缓冲缓冲

The solution contains 62.5 F. Tris PH8.3, 95 mM KC1, 3.8 mM

MgCl2, i2_5mM dithiothreitol and hydrazine 63% detergent; (b) taking the clarification liquid from the homogenized fish brain tissue of step (a); (4) adding specificity to the neuro virus with lice (d)) in the clarification solution, and carry out reverse transcription polymerase chain reaction; and

(4) The product of the obtained reverse transcription polymerase chain reaction is analyzed, and a nucleic acid fragment containing a neurovirus indicates that the fish body is infected with a neurovirus. The term "neurovirus" as used herein refers to a disease infecting the nervous system or invading the nervous system. Preferably, the neuroviral system is η(10) usnecrosis virus (NNV). Analysis by virus genomics 'Neuronectin virus is classified in Noda virus family (Ν.^仙十 contains «野田病毒属(4)Ph_daviruse(四)田病毒- γ(四): The former is infected with insects, the latter mainly infected with juveniles and young children under one month:=. The neuron necrosis virus is also referred to as a fish neuropathy virus or a fish encephalitis virus. The fish body suitable for use in the method of the present invention is any fish body. In a preferred embodiment of the present invention, the preparation is a total For the purpose of smoking 丄 坏死 坏死 坏死 1 1 1 1 ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 117 The method of the method of the present invention is to homogenize fish brain tissue in a lysis buffer. The acquisition of the fish brain tissue is obtained from the brain cavity of the fish body, and the embodiment thereof is the technology of the present invention. Those who have the usual knowledge in the field, for example, destroy the skull of the fish body and cut the fish brain tissue from the brain cavity.

One of the features of the present invention is that the fish brain is homogenized in the lysis buffer, and the lysis buffer can dissolve the fish brain tissue moderately, and expose the nucleic acid of the neuron to facilitate the subsequent reverse transcription polymerase chain reaction without completely From the fish brain. · 屯化神, 毋病毋 nucleic acid. The lysis buffer according to the present invention contains 62.5 mM Tris PH8.3 ^ 95 mM KC1 ^ 3.8 mM MgCl 2 ^ 12.5 mM - thiothreitol and ο·63% detergent, preferably, the detergent is Np 4 〇. The method for homogenizing the fish brain in the step (4) of the method of the present invention is such that the brain tissue is dispersed in the lysis buffer, so that the components in the buffer are fully reacted with the fish brain tissue to dissolve the fish brain moderately. Group ^ in the present invention, preferably when the time is good, (4) (4) digesting the fish brain according to the steps of the present invention, g- # ) 糸 from the homogenization of the fish brain tissue in the step Ο) The term "liquid" refers to the portion of the solution excluding the suspension and insoluble matter of the tissue (b), preferably, the fish brain tissue is homogenized in step H to obtain the clear liquid. Initiation: / (4) is a reverse transcription polymerase chain reaction in the I month liquid for the specific primer of the neurovirus. The primers according to the invention of the present invention are determinable based on the type of the neurovirus to be detected in the technical field to which the present invention pertains. Taking neuronecrosis virus as an example, it is a virus without a mantle, and has two poly A tails but a 5' cap (5'cap) single-stranded RNA with a molecular weight of 3.1. Kb (RNAl) and 1.4Kb (RNA2), in which RNA1 functions as RNA-dependent RNA polymerase, RNA2 can transduce the coat protein of the virus. In the process of necrosis and replication, the subgenomic RNA3 can be detected, sequenced and sequenced, and the sequence is identical to the 3-terminal sequence of RNA1, with an ORF, which can be translated into a non-structural protein. B2. In a preferred embodiment of the invention, step (c) is directed to the neuroviral-specific primer set for the RNA1, RNA2 or RNA3 region of the necrotic virus. Taking the RNA2 region as an example, Nishizawa et al. (Nishimura et al., 1994, Disease of Aquatic Organism 18: 103-107; Nishizawa et al., 1995, Journal of General Virology 76: 1563-1569) published two designs based on RNA2 sequences. The forward primer (FI, F2) and the three reverse primers (R1, R2, R3), through a combination of different arches and sub-arrays, carry out reverse transcription polymerase chain reaction, which can amplify five different size gene fragments (ΤΙ, T2, T3, T4, T5), this method can quickly and accurately detect the presence of a sample containing a small amount of virus; in the test of different fish species, the red spot grouper (five aAraara) and other fish species The reverse transcription polymerase chain reaction can only amplify two sets of nucleic acid fragments, T2 and T4, respectively, which show that the T2 and T4 fragments are highly retained on the neuronecrosis virus of different marine fish. In a more preferred embodiment of the invention, step (c) is directed to the neuroviral 117404.doc 丄 W6357-specific primer set for the region of the necrosis virus. As described in the following examples, the primer specific to the neuroviral in step (c) is preferably a primer comprising sequence identification number 1 and sequence identification number 2. The reverse transcription polymerase chain reaction according to the present invention is preferably an immediate reverse transcription polymerase chain reaction which can rapidly and quantitatively quantify the number of viruses using a small sample. According to the method of the present invention, it is preferred to further comprise the step (cl) of polymerase chain reaction using the product of the reverse transcription polymerase chain reaction of step (c) to further amplify the reaction product for profit detection. According to the step (d) of the present invention, the product of the resulting reverse transcription polymerase chain reaction is analyzed, and a nucleic acid fragment containing a neurovirus indicates that the fish body is infected with a neurovirus. According to a preferred embodiment of the invention, the nucleotide fragment of the neuroviral is RNA1, RNA2 or RNA3. In a preferred embodiment of the present invention, the step (^ and the reverse transcription polymerase chain reaction is carried out on a microfluidic wafer. The term "microfluidic wafer" as used herein refers to the detection process. The required components, such as the reaction tank, the heating reaction tank, the separation pipeline, and the detection tank, are concentrated on the same wafer, and then electrolyzed by an applied voltage, or by means of a miniaturized pump or centrifugal force. Driving samples or reagents in microchannels connected between components to complete detection. Microfluidic wafers are also referred to as "laboratory platform wafers." Biomedical detection or analysis using microfluidic wafers has experimental errors that reduce manual operations. It is another object of the present invention to provide a kit for detecting a fish infected nerve 117404.doc 12 virus, which comprises: The buffer contains 62.5 mM Tris pH 8.3, 95 mM KC1, 3.8 mMMgCl2, 12.5 mM dithiothreitol, and 0.63 ° / cleaning agent; And the reagents required for the reverse transcription polymerase chain reaction. Preferably, the buffer, dNTP and reverse transcriptase are included. The following examples are used to describe the invention in detail, but the invention is not limited thereto. [Examples] Example 1: Reverse transcription polymerase chain reaction detection of fish brain tissue was added with appropriate amount of lysis buffer (62.5 mM Tris pH 8.3, 95 mM KC1, 3.8 mM MgCl2, 12.5 mM dithiosulphate) The sugar alcohol and 0.63% NP-40) were ground and centrifuged, and the supernatant was taken. The supernatant was used as a template and the neuron necrosis virus specific primer pair (SEQ ID NO: 1 and 2) was reverse transcribed. Polymerase chain reaction. Fish brain tissue supernatant sample was taken 2 μΐ, 5 μΐ of MMLV reverse transcriptase polymerase buffer (5χ), specific primer pair 1 μl (10 μΜ), hydration to 23 μΐ, at 70 After 10 minutes of action at ° C, it was placed on ice for cooling; after adding 1 ml of dNTP (10 mM) and MMLV reverse transcriptase polymerase, it was allowed to act at 42 ° C for 60 minutes and then deactivated at 95 ° C for 15 minutes. 2 μΐ cDNA samples were added to the specific primers at both ends (10 μΜ) 1 μΐ, 10 times polymerase buffer 5 μΐ, polymerase 1 μΐ, 1 μΐ dNTP finally make up the secondary water to 50 μΐ and mix uniformly to carry out the reaction, the conditions are 94 ° C for 5 minutes, then at (94 ° C40 seconds, 55 ° C 40 seconds, 72 ° C 40 seconds) for 35 consecutive cycles, and finally 72t: 5 minutes to complete 117404.doc 13 1326357 reaction. Figure 1, it can be seen in the diseased fish sample, according to the present invention The method can be used for detecting (four) m-body wafers for reverse transcription polymerase chain reaction detection

The microfluidic wafer used in this example is shown in Fig. 2. The wafer contains three spaces 'replacement of a reverse transcription polymerase with a corresponding reagent and a polymerase chain reaction reagent respectively' and (4) computer setting and pump driving to enable detection of samples The polymerase chain reaction is continued after the reverse transcription polymerase chain reaction is completed, and finally the product is driven to capillary electrophoresis and analyzed. The detection method is as described in Example 1, except that the present example uses a microfluidic wafer for the reaction. In comparison, the sample 1 test took about 6 hours and 30 minutes, and this example took only 2 hours and 45 minutes. The results are shown in Fig. 3. It is understood that a fragment of 203 bp can be synthesized in the sample of the diseased fish sample, so that the method according to the present invention can be used for detecting the infection of the necrosis virus.

The results of electrophoresis analysis showed that a fragment of 203 bp, a necrosis virus infection, could be synthesized. Example 3: Microfluidic wafer for reverse transcription polymerase chain reaction detection. Quantitative line measurement and confidence test. For microfluidic wafer detection sensitivity, this example extracts RN A from purified necrosis virus and measures it with a spectrophotometer. The concentration is converted to the number of necrotic viruses and used to test the sensitivity of the microfluidic wafer. The GF-1 cell line (grouper fin cell line, bcrc No. 960094) was cultured at 28 ° C in L15 medium (GIBCOTM) containing 5% fetal bovine serum; the growth was increased to 8 densities, and washed with lxPBS. After 2 times of cells, add 1 ml of H7404.doc -14-1326357 virus solution. The control group was added with LBS5 culture medium without FBS and adsorbed for 28 minutes at 28C, then added 1〇mi containing 2 . /❶FBS culture medium continued to culture at 28 C for about 3 to 5 days. 9〇% of cells can be observed to produce cytopathic effect (CPE). At this time, the upper culture medium (ie, virus solution) is collected and scraped off. The cells were centrifuged at 50 〇〇 xg, 4 〇c for 1 minute, and the supernatant was slowly added to 2.2 °/ at 4 C. To NaCl, after completely dissolving, slowly add 5 〇 / 〇 of PEG 8000 (w / v) 'dissolved and placed at 4. Stir in the overnight refrigerator, centrifuge at 4t: looooxg for 1 hour, pour off the supernatant, and suspend the virus pellet in 2 ml of TES buffer _, store in -7 (TC refrigerator, and extract viral RNA, and Reverse transcription polymerase chain reaction was performed using a microfluidic wafer according to the method of Example 2. The viral RNA sequence was diluted to 100, 5, 25, and 12.5, and the microfluidic wafer was used for testing, and the detection intensity was recorded, and the experiment was repeated in three experiments. After that, the results are shown in Figure 4. The minimum detectable concentration is between 1〇_1〇〇 virus number', which is ten times higher than the general reverse transcription polymerase chain reaction method. Call for further understanding of microfluidic wafers in i 〇 1 1 是否 是否 是否 是否 是否 是否 是否 是否 ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' The number is proportional and credible (the results are shown in Figure 5). The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Modifications and changes are still not violated The spirit of the present invention should be as defined in the scope of the patent application described below. [Simple Description of the Scheme] 117404.doc 15 is the result of the reverse transcription polymerase chain reaction in Example 1 Figure 2 is a microfluidic wafer diagram The microfluidic wafer should be used for reverse transcription polymerase chain reaction. The microfluidic U-sheet is used for reverse transcription. The reverse linkage of the enzyme is shown in Figure 3. The microfluidic wafer is used for reverse transcription in the example 3. The detection should be performed. Known linkage reverse [simple description of component symbols] 2 4 5 polymerase chain reaction reagent polymerase chain reaction mixed reaction tank reverse transcription polymerase chain reaction reagent capillary electrophoresis tank pump I17404.doc 1326357 sequence table <11 〇> National success University <120> Method and kit for detecting fish body infected with neurovirus <130>None<160> 2 <170> Patentln version 3.2 <210> 1 <211> 19 <212> DNA < 213>Artificial sequence <400> 1 gacgcgcttc aagcaactc 19 <210> 2 <211> 12 <212> DNA <213> Artificial sequence <400> 2 cgaacactcc agcgacacag ta 22

117404.doc

Claims (1)

1326357 No. 0671 〇2772 Patent application t text application scope replacement (99 years March ^ _________ 10, t please special Wei Wei: 曰修8正1 | Announcement 1. Species detection fish body infection Noda The method of the neuroviral virus of the family Nodaviridae, which comprises: <k' (a) in a lysing buffer to homogenize fish brain tissue, wherein the lysing solution comprises 62.5 mM Tris pH 8 3 '95 mM κα, 3 8 福 g 】 2 12.5 mM dithiothreiol (DTT) and 0-63% detergent; (b) (c) (d) from the homogenized fish brain tissue in step (a) to clarify Liquid; Z-specific primer for the neuroviral is added to the clear liquid of step (8), and reverse transcription polymerase chain reaction is carried out; and the product of the reverse transcription polymerase chain reaction is analyzed, such as a nucleic acid fragment containing a neurovirus Indicates that the fish is infected with nerve poison. According to the method of claim 1, wherein the neuroviral system is neuronal necrosis virus (NNV). According to the method of claim 2, the fish system is selected from the group consisting of a money form, a sequel, a scorpion, and a blunt object. The method of claim 3, wherein the fish system is a grouper. According to the method of claim i, wherein the cleaning agent for the dissolution buffer is gland 40 ° 6. According to the method of claim 1, wherein step (a) homogenizes the fish brain tissue by grinding. Stomach 7. The method according to claim i, wherein the step (b) is to centrifuge the homogenized fish brain tissue to obtain the clear liquid. 1326357 Patent Application No. 096102772. Chinese Patent Application Substitute Replacement (March 99) 8. Method of root U item 2 'where step (4) is specific to the neuroviral specific primer for the nerve necrosis virus rna RNA or rnA3 area. Designed by the domain. .9. The method of Item 8 wherein step (4) is specific to the neuroviral virus and the primer is designed for the 譲2 region of the necrosis virus. 1 0. According to the method of claim 9, the step S (c) and the step (C) include the sequence identification number 1 and the sequence identification number 2 for the neuroviral vector. According to the method of claim 1, wherein the reverse transcription polymerase chain reaction in step (4) is an immediate reverse transcription polymerase chain reaction. According to the method of claim 1, further comprising the step (4) of performing a polymerase chain reaction using the product of the reverse transcription polymerase chain reaction of the step (4). 13. 14. According to the request! Or the method of 12, wherein the reverse transcription polymerase chain reaction in step (4) is carried out on a microfluidic wafer. A group of neuropathy 4 for detecting fish infection of the Luotian virus family, comprising: ” - The lysis buffer comprises 62.5 mM Tris pH 8 3, 95_Kd, 3.8 mMMgCl2, 12.5 touch dithiothreitol, and 〇63 % cleanser; a primer specific for the neuroviral; and a reagent required for a reverse transcription polymerase chain reaction. 15. The kit of claim 14 wherein the neuroviral system is a necrosis virus. The set of 15 wherein the fish system is selected from the patent application of 竣1, 326, 357 〇 〇 〇 〇 〇 〇 〇 • • • • • • • • ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 17. A group consisting of pure objects. 17. The kit according to claim 16, wherein the fish system is a grouper. / according to the set of claims, wherein the cleaning agent is Νρ·4 (^ _ 19. according to The kit of claim 15 wherein the primer specific for the neuroviral is set forth in the RNA1, 2 or Xie 3 region of the necrosis virus. 20. The kit according to claim 19, wherein the neuroviral is specific to the neuroviral Neuronal necrosis virus The RNA2 region is designed. According to the -month-seeking 2G set, the primers specific to the neurovirus include the sequence identification number 丨 and the sequence identification number 2 primer. 22. According to the set of claim 14, the other Contains - microfluidic wafers.