The complete genome sequence of hepatitis C virus in green turtles and its detection method

Abstract

The present invention provides a full genome sequence of hepatitis C virus of green turtles and a detection method thereof. The full genome sequence of the virus is 9430bp in length and has a GC content of 45%. The viral genome contains a long ORF with nucleotide positions at 517-9048. The precursor polyprotein encoded by the ORF is 2843aa in length. The full genome sequence, ORF sequence and amino acid sequence of the encoded protein of the green turtle hepatitis C virus are shown in SEQ ID NO:1-NO:3. According to the full genome sequence of the virus, 10 pairs of primers and 1 group of primer probes were designed for ordinary RT-PCR and fluorescent quantitative RT-PCR detection, respectively. Both methods have good specificity and reliable detection results. The detection method provided by the present invention can lay a solid foundation for the in-depth study of hepatitis C virus of green turtles and the epidemiological investigation of diseases, and can also standardize the release behavior of green turtles and improve the marine ecological environment.

Description

Technical Field

[0001] The invention relates to the technical field of molecular bioengineering, and in particular to a full genome sequence of hepatitis C virus of a green turtle and a detection method thereof. Background Art

[0002] The green turtle (Chelonia mydas) is a flagship marine species and umbrella species, and has always been regarded as a health indicator of the marine ecosystem. Due to long-term overexploitation, the green turtle has become an endangered species and is listed as a national first-class protected wild animal. In recent years, there have been continuous deaths of green turtles in breeding sites in Wenchang, Qionghai and other places in Hainan, and surviving individuals have also gradually become ill and died. The main symptoms of sick turtles are anorexia, mental depression, and slow reaction. An autopsy found that the livers of dead green turtles had obvious lesions. There are currently no reports of green turtles being infected with the hepatitis C virus. If the disease continues to spread and is transmitted to wild green turtle populations through human release and other behaviors, it will pose a great threat to marine ecological security. Summary of the invention

[0003] The first purpose of the present invention is to provide a complete genome sequence of the hepatitis C virus of green turtles to solve the current unclear technical problem of the hepatitis C virus that causes death to green turtles, thereby achieving the purpose of protecting turtles and improving marine ecology.

[0004] The second purpose of the present invention is to provide a method for detecting hepatitis C virus in green turtles to solve the current technical problem of difficulty in detecting the virus, thereby playing a role in early prevention of hepatitis C virus in green turtles.

[0005] The present invention provides a full genome sequence of green turtle hepatitis C virus, the full genome sequence of the virus is 9430bp in length, the GC content is 45%, the virus genome contains a long ORF, the nucleotide position is 517-9048, and the precursor polyprotein encoded by the ORF is 2843aa in length. The full genome sequence of green turtle hepatitis C virus, ORF sequence and encoded protein amino acid sequence are shown in SEQ ID NO:1-NO:3.

[0006] On the other hand, the present invention provides 10 pairs of primers and 1 set of primer probes for amplifying the whole genome sequence of the green turtle hepatitis C virus, which are used for conventional RT-PCR and fluorescent quantitative RT-PCR detection, respectively, and the sequences are as follows:

[0007] RT1269-1F:5'-GATCCGAGCGGCACCGTT-3'(SEQ ID NO:4)

[0008] RT1269-312R:5'-CAACTCGCCGTCCACTAGACTATT-3'(SEQ ID NO:5)

[0009] RT1269-15F:5'-CGTTCTAGAATGGGACAAATCGT-3'(SEQ ID NO:6)

[0010] RT1269-1240R:5'-CATCATCAAAAGCACCCATATACAT-3'(SEQ ID NO:7)

[0011] RT1269-1021F:5'-TTTTTAGAGCATATACCCCTGTCCAAT-3'(SEQ ID NO:8)

[0012] RT1269-2223R:5'-AGAAGCAAGTTTAAGGGCACATAGT-3'(SEQ ID NO:9)

[0013] RT1269-1976F:5'-TGATTATAGCCCTCCATGTTTATGTCAG-3'(SEQ ID NO:10)

[0014] RT1269-3184R:5'-GACATGAGCTATGAGCACACGTAAC-3'(SEQ ID NO:11)

[0015] RT1269-2986F:5'-GCTAATTGGACCCCTACGTCAC-3'(SEQ ID NO:12)

[0016] RT1269-4201R:5'-TAGGAATAACATCAATTGATAAACCTCT-3'(SEQ ID NO:13)

[0017] RT1269-3999F:5'-TAAAGACATCACCGAGGTGCCT-3'(SEQ ID NO:14)

[0018] RT1269-5207R:5'-GCTTGTACACCTGCTAACATTCG-3'(SEQ ID NO:15)

[0019] RT1269-4996F:5'-GCTGTAGATGCGGTTAAACAGTATG-3'(SEQ ID NO:16)

[0020] RT1269-6200R:5'-ATAACCTGTTAACCATATTGACTCATCC-3'(SEQ ID NO:17)

[0021] RT1269-6000F:5'-AGGGCTAATAACAATGCGAAAGT-3'(SEQ ID NO:18)

[0022] RT1269-7202R:5'-ACGCTTTCTTGCACTAACATTATCAT-3'(SEQ ID NO:19)

[0023] RT1269-6980F:5'-AACCCCCACCTAGTGTACTAAATGT-3'(SEQ ID NO:20)

[0024] RT1269-8271R:5'-ATCCAGTCTCTGGTCTATGTCATCTAC-3'(SEQ ID NO:21)

[0025] RT1269-8061F:5'-TCTCCTTGATAAGGAGAATAAACCACT-3'(SEQ ID NO:22)

[0026] RT1269-9430R:5'-TCCTCTTAGAAATTTTACGTCAATGC-3'(SEQ ID NO:23)

[0027] qF1: 5'-GCTCTTCTGGTTGTACTC-3' (SEQ ID NO: 24)

[0028] qR1: 5'-AGGGACTGGATCAAATTC-3' (SEQ ID NO: 25)

[0029] qP1: 5'-TTACTTACCAACACAGAGCATAGCACT-3' (SEQ ID NO: 26)

[0030] The present invention also provides a method for detecting hepatitis C virus in green turtles, that is, using the above detection primers to perform RT-PCR detection.

[0031] Preferably, the above detection method comprises the following steps:

[0032] (1) Extract RNA from tissues of green turtles suspected of being infected with the virus;

[0033] (2) synthesizing the first strand of cDNA from the final product RNA of step (1), and performing conventional qualitative RT-PCR amplification using the above-mentioned specific detection primers;

[0034] (3) Detection: Detect PCR product bands using 1.5% agarose gel electrophoresis, use 2 kb DNA marker as a fragment size control standard, and determine the detection results based on the electrophoresis diagram.

[0035] The PCR system in the above step (2) is as follows:

[0036]

[0037]

[0038]

[0039] On the other hand, the present invention also provides a method for detecting hepatitis C virus in green turtles, that is, using the above primer probe to perform fluorescent quantitative RT-PCR detection.

[0040] Preferably, the above detection method comprises the following steps:

[0041] (1) Extract RNA from tissues of green turtles suspected of being infected with the virus;

[0042] (2) synthesizing the first strand of cDNA from the final product RNA of step (1), and performing fluorescent quantitative RT-PCR amplification using the above-mentioned specific detection primer probe;

[0043] (3) Use a fluorescent quantitative PCR instrument to perform detection and determine the detection results based on the Ct value and amplification curve.

[0044] The PCR system in the above step (2) is as follows:

[0045]

[0046]

[0047]

[0048] The present invention uses bioinformatics technology to identify a new virus that can cause disease in green sea turtles. The virus belongs to the Flaviviridae family and the Hepacivirus genus (temporarily named Chelonia mydas Hepacivirus, ChHCV). A highly specific detection method is designed for the sequence of the virus, which plays a positive role in the prevention of hepatitis C in green sea turtles, can protect turtles, and contribute to the improvement of marine ecology. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a necropsy picture of a green sea turtle infected with hepatitis C virus;

[0050] Figure 2 This is a simplified diagram of the genome structure of the green turtle hepatitis C virus;

[0051] Figure 3 Phylogenetic analysis of green turtle hepatitis C virus and other viruses of the same genus;

[0052] Figure 4 This is the electrophoresis diagram of the virus detected by common RT-PCR;

[0053] Figure 5 This is the amplification curve of the virus detected by fluorescent quantitative RT-PCR. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0055] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to specific embodiments.

[0056] Example 1 Animal virus identification technology based on NGS sequencing

[0057] (1) Sample collection and RNA extraction: Before collecting green turtle tissue samples, the cryo-grinder should be precooled to the working state. After tissue collection, the samples should be immediately placed in a sterile EP tube containing one large and two small steel balls. Grind thoroughly for 1 min under refrigeration. Samples that are difficult to grind, such as intestines, can be ground repeatedly. After grinding, mark the samples and store them in a -80°C refrigerator.

[0058] The Magmax24 nucleic acid extractor from Thermo Fisher Scientific was used to extract nucleic acid from the samples. The reagent used was the MagMAX 96 Viral RNA Isolation Kit that came with the instrument to extract RNA from the samples. The specific steps are as follows:

[0059] Add 100 μL of the ground tissue homogenate to well A of the Magmax24 nucleic acid extractor and perform the extraction operation using the AM1836 program set on the instrument. After the program is completed, transfer the total nucleic acid obtained in well F to a new EP tube.

[0060] (2) NGS sequencing: The sample library was constructed using the MGI Easy-RNA Library Preparation Kit and sequenced using the MGI SEQ-200RS gene sequencer. The kit used for sequencing was the MGI SEQ-200RS High-throughput Sequencing Kit (FCL PE150), and the total running time was approximately 47 h.

[0061] (3) Sequencing data processing and analysis: The data finally generated by the MGI sequencing platform is a clean sequence with the identification QR codes at both ends removed, in fastp.gz format. After decompressing the data, the double-end data is obtained. The low-quality data is removed, and the sequences with read length less than 50bp, and the sequences containing too many abnormal bases or abnormal base percentages are removed. The virus-related sequences obtained by filtering are spliced ​​using Trinity software to obtain contigs using the laboratory data analysis platform, and the splicing is completed in combination with Megahit software. After removing the primer sequences of the RACE kit at both ends, the full genome sequence of the virus is obtained.

[0062] Example 2 Construction of virus evolution tree and verification of whole genome sequence

[0063] The virus was further subjected to phylogenetic analysis. The sequences of classic strains in Hepacivirus AN and "related but unclassified shark hepatitis C virus" used to construct the phylogenetic tree were downloaded from NCBI. The entire ORF coding region sequence was used for phylogenetic analysis to determine the genetic relationship of the new virus. The phylogenetic tree was constructed using Mega10.2 software, the maximum likelihood method, a bootstrap of 1000, and the GTR+G+I model. The results are shown in the figure. Figure 3 As shown, according to Figure 3 The results showed that the hepatitis C virus in green sea turtles was a new species of the genus Hepatitis C virus in the family Flaviviridae.

[0064] The whole genome sequence of the green turtle hepatitis C virus was further verified using the Sanger sequencing method, wherein a pair of primers was designed every 1000bp or so, and the primer sequences are shown in SEQ ID NO:4-NO:23. The whole genome sequence of the green turtle hepatitis C virus finally measured by Sanger sequencing is shown in SEQ ID NO:1. The sequencing result completed the 21 bases of the NGS sequencing result, and the consistency between the Sanger sequencing result and the NGS sequencing result was 99.78%.

[0065] Example 3 Bioinformatics analysis of the whole viral genome

[0066] The whole genome sequence obtained by Sanger sequencing was verified and analyzed using software such as DNAMAN to obtain basic data such as the sequence GC content. Then, the viral open reading frame (ORF) was searched online using tools such as RAST (Rapid Annotation using Subsystem Technology, http: / / rast.nmpdr.org) or NCBI ORF finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ). The obtained ORF sequence is shown in SEQ ID NO: 2.

[0067] The precursor polyprotein encoded by the virus ORF is 2843aa in length, and the amino acid sequence is shown in SEQ ID NO:1-NO:3. Protein Blast was performed on NCBI, and the most similar one was AWM96387.1, with a similarity of 86%. Among the species studied, the most similar ones were the hepatitis C viruses carried by the Chinese broad-headed pond turtle and the Chinese soft-shell turtle, with similarities of 75% and 68% respectively.

[0068] Example 4: Common RT-PCR Detection Method for Green Turtle Hepatitis C Virus

[0069] (1) Extract RNA from tissues of green turtles suspected of being infected with the virus (method as described above).

[0070] (2) The final product RNA of step (1) is subjected to cDNA first-strand synthesis, and ordinary qualitative RT-PCR amplification is performed using the specific detection primers. The primers can be selected from SEQ ID NO: 4-NO: 23, that is, any pair of 10 primers can be used for detection. The reaction system and conditions are as follows:

[0071]

[0072]

[0073] (3) Detect the PCR product bands by 1.5% agarose gel electrophoresis, use 2 kb DNA marker as the fragment size control standard, and determine the detection results based on the electrophoresis diagram.

[0074] This example uses 10 pairs of primers for detection, where "G1, P1-P9" are common RT-PCR reactions corresponding to the 10 pairs of primers. The detection results are as follows: Figure 4 As shown in the results, it can be seen that the detection primers of the present invention have strong specificity, the product bands are clearly visible, the detection results are reliable, and the detection of samples by ordinary RT-PCR method has stable and reliable performance.

[0075] Example 5 Fluorescence quantitative RT-PCR detection method for hepatitis C virus in green turtles

[0076] (1) Extract RNA from tissues of green turtles suspected of being infected with the virus (method as described above).

[0077] (2) The final product RNA of step (1) was subjected to cDNA first-strand synthesis, and fluorescent quantitative RT-PCR amplification was performed using the specific detection primer probe, and 1 μL (about 100 ng) of DNA of turtle herpesvirus type 5, sea bream iridovirus, and Vibrio parahaemolyticus were used as experimental references, respectively, and DEPC water was used as a negative control. The primer and probe sequences were shown in SEQ ID NO: 24-NO: 26, and the reaction system and conditions were as follows:

[0078]

[0079]

[0080]

[0081] (3) Use a fluorescent quantitative PCR instrument to perform detection and determine the detection results based on the Ct value and amplification curve.

[0082] In this example, a set of primers and probes were used to perform fluorescence quantitative RT-PCR detection. The detection results are shown in Figure 5 As shown in the results, it can be seen that the detection primer probe of the present invention has strong specificity, the positive sample amplifies a typical "S" curve, the detection result is reliable, and the fluorescent quantitative RT-PCR method also has a stable and reliable performance in detecting samples.

[0083] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. An amplification primer for amplifying the whole genome sequence of hepatitis C virus of green turtles, characterized in that: The full genome sequence of the green turtle hepatitis C virus is shown in SEQ ID NO: 1; the amplification primers are as follows: G1, including RT1269-1F and RT1269-312R, the sequence of RT1269-1F is shown in SEQ ID NO:4, and the sequence of RT1269-312R is shown in SEQ ID NO:5; P1, including RT1269-15F and RT1269-1240R, the sequence of RT1269-15F is shown in SEQ ID NO:6, and the sequence of RT1269-1240R is shown in SEQ ID NO:7; P2, including RT1269-1021F and RT1269-2223R, the sequence of RT1269-1021F is shown in SEQ ID NO:8, and the sequence of RT1269-2223R is shown in SEQ ID NO:9; P3, including RT1269-1976F and RT1269-3184R, the sequence of RT1269-1976F is shown in SEQ ID NO: 10, and the sequence of RT1269-3184R is shown in SEQ ID NO: 11; P4, including RT1269-2986F and RT1269-4201R, the sequence of RT1269-2986F is shown in SEQ ID NO: 12, and the sequence of RT1269-4201R is shown in SEQ ID NO: 13; P5, including RT1269-3999F and RT1269-5207R, the sequence of RT1269-3999F is shown in SEQ ID NO: 14, and the sequence of RT1269-5207R is shown in SEQ ID NO: 15; P6, including RT1269-4996F and RT1269-6200R, the sequence of RT1269-4996F is shown in SEQ ID NO: 16, and the sequence of RT1269-6200R is shown in SEQ ID NO: 17; P7, including RT1269-6000F and RT1269-7202R, the sequence of RT1269-6000F is shown in SEQ ID NO: 18, and the sequence of RT1269-7202R is shown in SEQ ID NO: 19; P8, including RT1269-6980F and RT1269-8271R, the sequence of RT1269-6980F is shown in SEQ ID NO:20, and the sequence of RT1269-8271R is shown in SEQ ID NO:21; P9, including RT1269-8061F and RT1269-9430R, the sequence of RT1269-8061F is shown in SEQ ID NO:22, and the sequence of RT1269-9430R is shown in SEQ ID NO:

23.

2. An amplification primer probe for amplifying the whole genome sequence of green turtle hepatitis C virus, characterized in that: The full genome sequence of the green turtle hepatitis C virus is shown in SEQ ID NO: 1; the sequences of the amplification primers and probes are as follows: qF1, the sequence of which is shown in SEQ ID NO:24; qR1, the sequence of which is shown in SEQ ID NO:25; qP1, the sequence of which is shown in SEQ ID NO:26.

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