CN102559484B - Prawn infectivity muscle necrosis virus fluorescent quantificationally PCR detecting kit and detection method - Google Patents

Abstract

The invention relates to a detection kit and a detection method for shrimp infectious myonecrosis virus, belonging to the technical field of detection of marine biological pathogens. Including the box body, the tube hole bracket is arranged in the box body, and the following tubes are placed on the tube hole bracket: (1), fluorescent quantitative PCR reaction solution tube; forward primer sequence; reverse primer sequence; TaqMan probe sequence; (2) ), deionized water tubes, containing sterile deionized water; (3), negative control tubes, containing SPF (specific pathogen-free) prawn nucleic acid; (4), standard quality tubes, containing infectious myonecrosis virus 10 ⁷ , 10 ⁶ ,10 ⁵ ,10 ⁴ ,10 ³ ,10 ² ,10,1 copy cDNA positive clones. The invention has high popularization and application value, and the detection method is sensitive, specific and easy to operate.

Description

Fluorescent quantitative PCR detection kit and detection method for shrimp infectious myonecrosis virus

technical field

The invention relates to a detection kit and a detection method for shrimp infectious myonecrosis virus, belonging to the technical field of detection of marine biological pathogens.

Background technique

Infectious myonecrosis is a new viral infectious disease that endangers the production of prawns discovered in Brazil in 2002. In 2004, Dr. LightnerDV of the University of Arizona found that the disease was caused by a new virus and named it Infectious myonecrosis virus (IMNV for short). The novel virus is a double-stranded RNA virus belonging to the family Holoviridae. The susceptible shrimp species of shrimp infectious myonecrosis virus is Penaeus vannamei, mainly juvenile shrimp infected 60-80 days old. The virus can cause muscle tissue necrosis in the whole body of infected prawns. Under normal circumstances, pathological death occurs slowly and the mortality rate is not high, but there are deaths throughout the breeding process, and the cumulative mortality rate can reach 70%. The virus has now spread to Asia and is threatening the culture of Penaeus vannamei in China.

In 2007, AndradeTPD et al. established a fluorescent quantitative PCR detection method for IMNV, which can detect at least 10 copies of IMNV. The sensitivity of this detection method needs to be further improved. Since the fluorescent quantitative PCR detection method is an important and effective method for the quantification and early prevention of shrimp viruses, it is extremely important to establish a highly sensitive fluorescent quantitative PCR detection method and a virus detection kit used in conjunction with the detection method.

Contents of the invention

The purpose of the present invention is to solve the problem that the sensitivity of existing fluorescent quantitative PCR detection method is not high, provide the fluorescent quantitative PCR detection kit of prawn infectious myonecrosis virus, also provide the detection method that utilizes this kit to detect simultaneously, The detection method is sensitive, specific and easy to operate.

The present invention is achieved through the following technical solutions:

The fluorescent quantitative PCR detection kit for shrimp infectious myonecrosis virus comprises a box body 1, and a tube hole bracket 5 is arranged in the box body 1. The special feature is that the tube hole bracket 5 is provided with the following pipe fittings:

(1) Fluorescent quantitative PCR reaction solution tube 6, which contains 2× fluorescent quantitative PCR buffer, a mixture of positive and negative primers for shrimp infectious myonecrosis virus and fluorescently labeled TaqMan probes;

Forward primer sequence: 5'-GATGGCAGCACGTCAAAATG-3';

Reverse primer sequence: 5'-CAGCTGTTCCTGGGACTTCCT-3';

TaqMan probe sequence: 5'-CTATTTCCTCCACTAATTCAGAGC-3', the 5' end of the probe sequence is labeled with FAM, and the 3' end is labeled with TAMRA;

(2), deionized water pipe 3, filled with sterile deionized water;

(3) Negative control tube 4, containing SPF (specific pathogen free) prawn nucleic acid;

(4) Standard tube 2, containing 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10,1 copies of cDNA positive clones of infectious myonecrosis virus respectively.

The fluorescent quantitative PCR detection method of shrimp infectious myonecrosis virus is as follows:

(1) Fluorescent quantitative PCR reaction system:

Reagent Volume/system (μl) Final concentration Fluorescent quantitative PCR reaction solution 11.2 1×real-time PCR buffer, 0.2μM forward and reverse primers, 0.4μM probe Standard or template cDNA 1 sterile deionized water 7.8 total capacity 20 -

(2) Fluorescent quantitative PCR reaction:

Denaturation at 95°C for 30s, 1 cycle;

Denaturation at 95°C for 5s, annealing and extension at 62°C for 20s, 40 cycles.

The fluorescent quantitative PCR detection kit for shrimp infectious myonecrosis virus of the present invention redesigns primers and probes according to the entire gene sequence of infectious myonecrosis virus, uses fluorescent quantitative PCR to detect the fluorescence intensity of the labeled probe in real time, and determines it according to the analysis software The virus content of the sample, this kit can be applied to quantitative research on the proliferation of infectious myonecrosis virus in prawns, the detection of infectious myonecrosis virus in the process of cultivating healthy shrimp seedlings, etc., and has high promotion and application value. The method is sensitive, specific and easy to operate.

Description of drawings

Figure 1: Schematic diagram of the detection sensitivity of shrimp infectious myonecrosis virus fluorescent quantitative PCR;

(a) Detection sensitivity of fluorescent quantitative PCR, 10 ⁷ -1 copies of infectious myonecrosis virus cDNA all exceeded the threshold; (b) Standard curve of fluorescent quantitative PCR detection of infectious myonecrosis virus;

Figure 2: Schematic diagram of specific detection of shrimp infectious myonecrosis virus by fluorescent quantitative PCR;

Figure 3: Schematic diagram of the structure of the fluorescent quantitative PCR detection kit for shrimp infectious myonecrosis virus.

Detailed ways

The specific embodiments of the present invention are given below with reference to the accompanying drawings, which are used to further describe the present invention.

Example 1

The fluorescent quantitative PCR detection kit for shrimp infectious myonecrosis virus comprises a box body 1, and a tube hole support 5 is arranged in the box body 1, and the following pipe fittings are arranged on the tube hole support 5:

(1) Fluorescent quantitative PCR reaction solution tube 6, which contains 2× fluorescent quantitative PCR buffer, a mixture of positive and negative primers for shrimp infectious myonecrosis virus and fluorescently labeled TaqMan probes;

Forward primer sequence: 5'-GATGGCAGCACGTCAAAATG-3';

Reverse primer sequence: 5'-CAGCTGTTCCTGGGACTTCCT-3';

TaqMan probe sequence: 5'-CTATTTCCTCCACTAATTCAGAGC-3', the 5' end of the probe sequence is labeled with FAM, and the 3' end is labeled with TAMRA;

(2), deionized water pipe 3, filled with sterile deionized water;

(3) Negative control tube 4, containing SPF (specific pathogen free) prawn nucleic acid;

(4) Standard tube 2, containing 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10,1 copies of cDNA positive clones of infectious myonecrosis virus respectively.

The fluorescent quantitative PCR detection method of shrimp infectious myonecrosis virus is as follows:

(1) Fluorescent quantitative PCR reaction system:

Reagent Volume/system (μl) Final concentration Fluorescent quantitative PCR reaction solution 11.2 1×real-time PCR buffer, 0.2μM forward and reverse primers, 0.4μM probe Standard or template cDNA 1 sterile deionized water 7.8 total capacity 20 -

(2) Fluorescent quantitative PCR reaction:

Denaturation at 95°C for 30s, 1 cycle;

Denaturation at 95°C for 5s, annealing and extension at 62°C for 20s, 40 cycles.

The detection process can be seen intuitively through the accompanying drawings 1-2, wherein (a) in Figure 1 is the detection sensitivity of fluorescent quantitative PCR, and 10 ⁷ -1 copies of infectious myonecrosis virus cDNA all exceed the threshold, and in Figure 2 Above the threshold are 10 ⁶ and 10 ³ copies of infectious myonecrosis virus cDNA, and the curves below the threshold are DNA of white spot syndrome virus, DNA of infectious subcutaneous and hematopoietic tissue necrosis virus, DNA of hepatopancreas parvovirus, DNA of Penaeus monodon Symptovirus DNA, SPF shrimp DNA, sterile water.

Advantages of the method: (1) High sensitivity: 1 copy of infectious myonecrosis virus can be detected. (2) Good specificity: no crossover with other 4 common shrimp viruses and shrimp tissues in Figure 2

reaction.

(3) Simple and rapid operation: the positive and negative primers and probes of infectious myonecrosis virus are added to the fluorescent quantitative PCR reaction solution, which simplifies the operation steps.

Claims (1)

1. A fluorescent quantitative PCR detection kit for shrimp infectious myonecrosis virus, comprising a box body (1) with a tube hole bracket (5) inside the box body (1), which is characterized in that the tube hole bracket (5) is placed with The following fittings: 1) Fluorescent quantitative PCR reaction solution tube (6), which contains 2× fluorescent quantitative PCR buffer, a mixture of positive and negative primers for shrimp infectious myonecrosis virus and fluorescently labeled TaqMan probes; Forward primer sequence: 5'-GATGGCAGCACGTCAAAATG-3'; Reverse primer sequence: 5'-CAGCTGTTCCTGGGACTTCCT-3'; TaqMan probe sequence: 5'-CTATTTCCTCCACTAATTCAGAGC-3', the 5' end of the probe sequence is labeled with FAM, and the 3' end is labeled with TAMRA; 2), deionized water pipe (3), filled with sterile deionized water; 3) Negative control tube (4), containing SPF prawn nucleic acid; 4) Standard quality tube (2), containing 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10,1 copies of positive cDNA clones of infectious myonecrosis virus respectively.