Yellow fever virus detection kit based on RPA technology and applications of yellow fever virus detection kit

Abstract

The invention discloses a yellow fever virus detection kit based on the RPA technology and applications of the yellow fever virus detection kit. The experiment proves that an RPA primer and a probe of the yellow fever virus can effectively amplify a target gene, the specificity achieves 100%, the sensitivity is 1x10<2> copies / reaction, the virus titer capable of being detected in a clinical simulation sample is 10<2>TCID50 / mL, and the sensitivity is equivalent to that of the fluorescent quantitative PCR; no cross reaction with the congeneric dengue virus, encephalitis B virus and tick-borne encephalitis virus nucleic acid exists. For the RPA isothermal amplification system provided by the invention, the reaction is rapid, the temperature range is wide, under the temperature of 37-42 DEG C, the effective amplification of the target gene can be realized, the yellow fever virus detection kit can be used for the on-site rapid detection for nucleic acid infected with the yellow fever virus, and meanwhile, an available rapid detection method is provided for the on-site screening of pathogenes. The yellow fever virus detection kit has great significance for preventing the infection and propagation of the yellow fever virus in China, and for the inspection and quarantine of epidemic areas and entry and exit ports.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a yellow fever virus detection kit based on RPA technology and an application thereof. Background technique

[0002] Yellow fever virus (Yellow fever virus) belongs to the Flavivirus genus of the Flaviviridae family, can cause severe hemorrhagic fever symptoms, is highly contagious, and has a case fatality rate of 23%. ～ 90%. The main manifestations are acute fever accompanied by severe bleeding. Yellow fever is mainly prevalent in Africa. China is not an endemic area for yellow fever virus. However, with the rapid development of global trade and tourism, human exchanges between different countries and regions have become increasingly frequent. Many infectious diseases have broken the borders of regions and countries. The scope of transmission is becoming wider and wider, and our country has also faced the threat of imported infectious diseases many times. In March 201...

Examples

Embodiment 1

[0066] The RPA detection primer of embodiment 1, yellow fever virus and probe and detection method thereof

[0067] 1. Design and synthesis of primers and probes for RPA detection of yellow fever virus

[0068] Download the whole gene sequence of yellow fever virus, use DNASTAR.Lasergene.v7.1 software for sequence comparison analysis, select the nucleoprotein (NP) gene segment (166bp in length) that is conserved within the species and specific among the species as the detection target gene, Design the RPA detection primer and probe of yellow fever virus according to following design requirements: (1) RPA primer length is 30-35 bases, and GC content is between 40-60%, avoids forming secondary structure and hairpin structure; Primer Special sequences such as long strings of polypurine or polypyrimidine should be avoided; the first 3-5 nucleotides at the 5' end should avoid polyguanine, preferably cytosine; (2) The length of the RPA probe is 46- 52 bases, inserting THF (tetrahyd...

Embodiment 2

[0081] The sensitivity comparison of the RPA detection of embodiment 2, yellow fever virus and Real-time PCR detection

[0082] 1. Yellow fever virus RPA detection

[0083] Adopt the RPA detection method of yellow fever virus in the step 2 of embodiment 1, use the yellow fever pseudovirus RNA solution of 10 times gradient dilution as template (the amount of template added is respectively: 10 5 copies / reaction, 10 4 copies / reaction, 10 3 copies / reaction, 10 2 copies / reaction, 10copies / reaction) for RPA amplification.

[0084] 2. Real-time PCR detection of yellow fever virus

[0085] The RNA solution of yellow fever pseudovirus with 10 times gradient dilution is used as template (the amount of template added is respectively: 10 5 copies / reaction, 10 4 copies / reaction, 10 3 copies / reaction, 10 2 copies / reaction, 10copies / reaction), using the primers and probes in Table 1, using the one-step real time RT-PCR kit (Takara, DRR064A) for Real-time PCR, the reaction system in e...

Embodiment 3

[0098] The specific detection of the RPA primer of embodiment 3, yellow fever virus and probe

[0099] They belonged to yellow fever virus 17D (YFV-17D, yellow fever pseudovirus), dengue virus type 1 (DEN-1) GZ01 strain, Japanese encephalitis virus (JEV) SA14-14-2 strain, tick-borne encephalitis The viral nucleic acid RNA of the virus (TBEV) Senzhang strain was used as a template, and the RPA detection method of yellow fever virus in step 2 of Example 1 was used to detect the specificity of the RPA primers and probes of the present invention.

[0100] The result is as figure 2 shown. The experimental results show that the RPA primer probes can specifically amplify the target gene and have no cross-reaction with the nucleic acids of three other viruses of the same genus (dengue virus type 1, Japanese encephalitis virus, and tick-borne encephalitis virus), indicating that The RPA primer and probe of the yellow fever virus of the present invention have better specificity.

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a yellow fever virus detection kit based on RPA technology and an application thereof. Background technique

[0002] Yellow fever virus (Yellow fever virus) belongs to the Flavivirus genus of the Flaviviridae family, can cause severe hemorrhagic fever symptoms, is highly contagious, and has a case fatality rate of 23%. ～ 90%. The main manifestations are acute fever accompanied by severe bleeding. Yellow fever is mainly prevalent in Africa. China is not an endemic area for yellow fever virus. However, with the rapid development of global trade and tourism, human exchanges between different countries and regions have become increasingly frequent. Many infectious diseases have broken the borders of regions and countries. The scope of transmission is becoming wider and wider, and our country has also faced the threat of imported infectious diseases many times. In March 201...

Examples

Embodiment 1

[0066] The RPA detection primer of embodiment 1, yellow fever virus and probe and detection method thereof

[0067] 1. Design and synthesis of primers and probes for RPA detection of yellow fever virus

[0068] Download the whole gene sequence of yellow fever virus, use DNASTAR.Lasergene.v7.1 software for sequence comparison analysis, select the nucleoprotein (NP) gene segment (166bp in length) that is conserved within the species and specific among the species as the detection target gene, Design the RPA detection primer and probe of yellow fever virus according to following design requirements: (1) RPA primer length is 30-35 bases, and GC content is between 40-60%, avoids forming secondary structure and hairpin structure; Primer Special sequences such as long strings of polypurine or polypyrimidine should be avoided; the first 3-5 nucleotides at the 5' end should avoid polyguanine, preferably cytosine; (2) The length of the RPA probe is 46- 52 bases, inserting THF (tetrahyd...

Embodiment 2

[0081] The sensitivity comparison of the RPA detection of embodiment 2, yellow fever virus and Real-time PCR detection

[0082] 1. Yellow fever virus RPA detection

[0083] Adopt the RPA detection method of yellow fever virus in the step 2 of embodiment 1, use the yellow fever pseudovirus RNA solution of 10 times gradient dilution as template (the amount of template added is respectively: 10 5 copies / reaction, 10 4 copies / reaction, 10 3 copies / reaction, 10 2 copies / reaction, 10copies / reaction) for RPA amplification.

[0084] 2. Real-time PCR detection of yellow fever virus

[0085] The RNA solution of yellow fever pseudovirus with 10 times gradient dilution is used as template (the amount of template added is respectively: 10 5 copies / reaction, 10 4 copies / reaction, 10 3 copies / reaction, 10 2 copies / reaction, 10copies / reaction), using the primers and probes in Table 1, using the one-step real time RT-PCR kit (Takara, DRR064A) for Real-time PCR, the reaction system in e...

Embodiment 3

[0098] The specific detection of the RPA primer of embodiment 3, yellow fever virus and probe

[0099] They belonged to yellow fever virus 17D (YFV-17D, yellow fever pseudovirus), dengue virus type 1 (DEN-1) GZ01 strain, Japanese encephalitis virus (JEV) SA14-14-2 strain, tick-borne encephalitis The viral nucleic acid RNA of the virus (TBEV) Senzhang strain was used as a template, and the RPA detection method of yellow fever virus in step 2 of Example 1 was used to detect the specificity of the RPA primers and probes of the present invention.

[0100] The result is as figure 2 shown. The experimental results show that the RPA primer probes can specifically amplify the target gene and have no cross-reaction with the nucleic acids of three other viruses of the same genus (dengue virus type 1, Japanese encephalitis virus, and tick-borne encephalitis virus), indicating that The RPA primer and probe of the yellow fever virus of the present invention have better specificity.

Images