TH7610C3 - How to detect macrobraciam virus Rosenbeji Ayanoidavirus (Macrobrachium rosenbergii nodavirus, MrNV) and the aforementioned diagnostic kits. - Google Patents

Abstract

DC60 (21/09/55) This invention is related to the detection of the macrobracium virus. Macrobrachium rosenbergii nodavirus (MrNV), a disease that causes the muscles of post larvae to turbulent white shrimp (post larvae) do not invert and die in shrimp and shrimp. Mature By this invention provided Two pairs of new primers were created, one of which checked for the macrobracic virus. Rosenbeji Eye Danovirus together with an already revealed primer With the reverse technique A 2-step PCR transcript utilizing a single tube nested PCR technique, a primer built. The remaining pairs control the internal reaction of the PCR. Which the virus detection method by PCR in conjunction with the use The primer mentioned here can increase sensitivity. The accuracy of the examination can be improved. And reduce the chance of Less contaminated with other infectious agents In less time Using the reverse method Transcript A single PCR or a two-tube reversible PCR technique uses two PCR tubes (two tube nested PCR technique). Macrobrachium virus Rosenbeji Ayadanovirus was raised for convenience. Fast to detect and cheap to detect Mac Cobra Chiam virus. Rosenbeji Ayanoidavirus is essential and essential to the lobster breeder's production system, lobster farming, and the verification of lobster products imported and exported abroad. The invention relates to the diagnosis of macrophages. Macrobrachium rosenbergil nodavirus (MrNV), a disease that causes the muscles of post larvae to turbid white shrimp (post larvae), do not invert and die in young shrimp and shrimp. Mature By arranging to create a prime McBruchiam's Mercedes Rosenberger Eyedanovirus and it is applied in reverse. A 2-step PCR transcript using a single tube nested PCR technique offers greater sensitivity and accuracy. And takes less time Using the reverse method Transcript One-step PCR or two tube nested PCR technique, 2-step reverse transcript or two-tube PCR technique has also been produced. Macrobraciam The Rosenbeji Eyedanovirus was established for the convenience, speed of detection and low cost, with the diagnosis of Macrobrachiamine Rosenburg Ayanoidavirus is vital and essential to the paternity production system. Shrimp breeder Shrimp farming as well as verification of shrimp products that are imported and exported abroad.

Claims (8)

1. Primers (2 pairs) used for diagnosing macrobracium Rosenbergiaanoda virus (Macrobrachium rosenbergil nodavirus, MrNV). Which consists of the nucleotide sequence as follows: MrNV-FN 5 \ '- GAT GTC GAT CAG TTA CTT AGG C-3 \' MrNV-RN 5 \ '- GTG AAA CTT CCA CTG GTG TAG-3 \' MrNV18S-F 5 \ '- GAG CCA GAG GTA ATG ATA AAG A-3 \' MrNV18S-R 5 \ '- GCT CTT AAT CTG TCA ATC CTT C-3 \' 2. Diagnosis of macrophages virus Rosenberger Eainoda virus by reverse method A 2-step PCR transcript using a single tube nested PCR technique using a primer from claim 1 follows these steps: 2.1 Prepare the sample. Shrimp that need to be tested for Macrobrachium virus Rosenberger Eynodavirus is extracted from RNA 2.2 DNA from 2.1 is used to detect the virus through the reverse process. First step RT-PCR 2.3 The product obtained from the PCR process, step one in item 2.2, is used to continue the reaction in Reverse process Transcript Nested step PCR 2.4 The PCR yield obtained from clause 2.3 was examined by using gel electrophoresis technique. 3. Macrobrachium diagnosis Rosenborg Ainodavirus according to claim 2 where the reaction of the reverse process PCR transcript, step one The reaction component is 2X PCR buffer 10 μl (muL) 1 mu M Mr18S-F 0.2 ml (mL) 1 mu M Mr18S-R 0.2 ml (mL) 1 mu M MrNV-F 0.5 ml (mL) ) 1 mu M MrNV-R 0.5 ml (mL) RT / Tag Supercript lll 0.5 μl (mL) Template 2.0 μl (muL) Distilled water (DW) 1.1 μl (muL) Total 15.0 μl (muL ) The PCR reaction was 94 ° c. 2 min. 94 ° c. 20 sec. 55 °. 30 sec. 15 cycles. 72 ํ. 30 sec. 72 ํ. 2 min. 20 ํ. Oc. 4. Macrobrachium diagnosis Rosenborg Ainodavirus according to claim 2 where the reaction of the reverse process Second-step PCR transcript It has reaction components: 10 x PCR buffer 2.5 mu L 50 m M MgCl2 0.75 m L 10 m M dNTPs 0.5 m L 10 m M Mr18S-F 0.1 mu L 10 mu M Mr18S-R 0.1 mu L 10 mu M MrNV-FN 0.4 mu L 10 mu M MrNV-RN 0.4 mu L 5 Unit Tag DNA Polymerase 0.25 mu L Distilled water (DDW) 10.0 mu L First step PCR 15.0 m L Total 30.0 m L Reaction The PCR used is 94 ํ. 2 min. 94 ํ. 20 sec., 50 ํ. 30 sec., 30 cycles. 72 ํ. 30 sec., 72 ํ. 2 min. 20 ํ. Oc. 5. Macrobrachium diagnosis Rosenborg Ainodavirus, pursuant to any of the 2nd to 4th claim, where the produce obtained from the reverse process Transcript PCR in case of shrimp infected with macrophages The Rosenberger Ayanoid virus is found to be productive in both the first stage of the PCR process and the second stage of the PCR process. 6. Macrobrachium diagnosis Rosenborg Ainodavirus under any of the 2nd to 4th claim, where the produce obtained from the reverse process Transcript PCR in case of shrimp infected with macrophages Rosenberger Ainoda virus is severe only to be productive in second-stage PCR process. 7. Macrobrachium diagnosis Rosenberger Ayanoidavirus according to Claim 2 - 6, any one applies to lobsters. 8. Macrobraciam Diagnostic Kit Rosenbeji Ayano Virus by means of claim 2 - 4, any of which includes, but is not limited to: 1 mu M of the MrNV-F primer, MrNV primer. -R, MrNV18S-F primer, MrNV18S-R 10mM primer of MrNV-FN primer, MrNV-RN primer, MrNV18S- primer F and MrNV18S-R primer 1.5 mM MgCl2, 10mM dNTP and a positive control.