CN102618531A - Method for confirming 3.-terminus sequence of virus RNA (Ribonucleic Acid) molecule - Google Patents

Abstract

A method for determining the 3' end sequence of a viral RNA molecule, comprising the steps of extracting viral RNA, purifying the tailed viral RNA, reverse transcription reaction of the tailed viral RNA, and polymerase chain reaction amplification of the viral reverse transcription product. The terminal transfer reaction of viral RNA After adding the tail through the terminal transfer reaction, the reverse transcription reaction can be used to accurately determine the 3' terminal sequence of the viral RNA molecule, which is convenient and quick. Since Oligo dT or modified Oligo dT primers can be used for reverse transcription reaction, the process of designing and synthesizing specific primers for a specific viral RNA molecule is omitted, which greatly simplifies the experimental cycle and saves experimental costs. At the same time, the present invention can also be applied to the determination of the 3' end sequence of other RNA molecules that do not have a PolyA tail structure at the 3' end.

Description

Method for determining the 3' end sequence of viral RNA molecule

technical field

The invention provides a method for quickly and accurately determining the 3' end sequence of a viral RNA molecule.

Background technique

Viruses are non-cellular organisms composed of nucleic acids and proteins, strictly parasitic in cells, and use the host's enzyme system to replicate. Relying on this mechanism, the virus can lead to the occurrence of persistent infection or the transformation of cells to form tumors. At present, the research on viruses mainly focuses on three aspects: on the one hand, to effectively prevent and control the occurrence and prevalence of various viral diseases; on the other hand, to further understand the basic problems of living matter through the understanding of viruses; The three aspects are to use viruses to benefit mankind. But no matter which aspect, the study of viral genetic material is particularly important.

A virus has only one specific type of nucleic acid, either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). For DNA viruses, polymerase chain reaction is currently used to achieve a large amount of amplification of viral DNA sequences in vitro, so that the viral DNA genome can be sequenced, analyzed and other related work to understand the genetic information of the virus. When studying RNA viruses, it is also necessary to determine the sequence of the RNA virus genome. However, polymerase chain reaction can only amplify DNA strands, and cannot directly amplify RNA strands. Therefore, under normal circumstances, it is necessary to reverse-transcribe the viral RNA into the corresponding first strand of cDNA, then clone the reverse-transcribed product by polymerase chain reaction, and determine the sequence of the cDNA to obtain the sequence of the corresponding viral RNA. The genome of some RNA viruses is the same as the messenger RNA (mRNA) of eukaryotic cells. The 3' end of the molecule has a polyA tail (polyA) structure, generally 40-200 polyA nucleotides. Therefore, this characteristic can be used to design reverse transcription primers, commonly used oligo-T nucleotide Oligo dT primers or modified Oligo dT primers (adding a nucleotide sequence at the 5' end of Oligo dT), through Oligo dT and The polyA at the 3' end of the viral RNA is paired to complete the reverse transcription reaction. After polymerase chain reaction amplification and sequence determination, the sequences of the 3' ends of these RNA virus genomes can be deduced. However, the 3' end of the genome of other RNA viruses does not have a polyA structure. For these viral RNA sequences, it is not possible to directly use Oligo dT primers or modified Oligo dT primers for reverse transcription. The traditional method is to utilize random primers to bind to the 3' end of the viral RNA molecule to carry out the reverse transcription reaction. Since random primers can only randomly combine with the 3' end of the viral RNA molecule and extend toward the 5' end of the viral RNA molecule, this method can only accurately locate the 5' end of the viral RNA molecule, thereby obtaining the 5' end of the viral RNA molecule. end sequence. Since the 3' end of the viral RNA molecule is combined with a random primer (not necessarily bound to the 3' end), it cannot be accurately positioned, and it is relatively difficult to obtain the sequence of the 3' end of the viral RNA molecule.

Terminal transferase is a template-free DNA polymerase that catalyzes the incorporation of deoxynucleotides into the 3' hydroxyl terminus of DNA molecules. Single- and double-stranded DNA molecules with protruding, recessed, or blunt ends can serve as substrates for terminal transferase. In addition, terminal transferase also has the activity of catalyzing the binding of deoxynucleotides to the 3' hydroxyl end of RNA molecules. Therefore, using this characteristic of terminal transferase, 10 to 50 oligo-A deoxynucleotides are added to the 3' end of the viral RNA molecule to process the general viral RNA into a hybrid of RNA and DNA with a polyA tail at the 3' end. The sequence of the 3' end of the viral RNA molecule can be accurately obtained by using Oligo dT or modified Oligo dT primers for reverse transcription reaction.

In addition to messenger RNA, RNA also exists in the form of ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA) and small nuclear RNA (snRNA), all of which have important biological functions. Since a small number of messenger RNAs and other types of RNA molecules do not have a polyA structure, the sequences of these RNAs cannot be directly obtained by reverse transcription using Oligo dT primers or modified Oligo dT primers. Therefore, it is also possible to use terminal transferase to add 10 to 50 oligo-A deoxynucleotides at the 3' end of the RNA molecule to process general RNA into a hybrid molecule of RNA and DNA with a PolyA tail at the 3' end. , and then use Oligo dT or modified Oligo dT primers for reverse transcription reaction, the sequence of the 3' end of the RNA molecule can be accurately obtained.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for quickly and accurately determining the 3' end sequence of the viral RNA molecule.

The technical solution adopted to solve the above technical problems comprises the following steps:

1. Extraction of viral RNA

Viral RNA was extracted by conventional methods, the quality of viral RNA was detected, and the concentration of viral RNA was quantified to 1 μg/μL.

2. Terminal transfer reaction of viral RNA

Add viral RNA, deoxyadenosine triphosphate and diethyl pyrocarbonate-treated water at a concentration of 10mmol/L to 12.5μL, and 10mmol/L deoxyadenosine triphosphate and diethylpyrocarbonate-treated water, virus The volume ratio of the RNA is 1:4:7.5, mix well, incubate at 70°C for 15 minutes to denature the viral RNA, obtain the denatured viral RNA solution, put it on ice for 2-10 minutes, and add it to the centrifuge tube containing the denatured viral RNA solution Terminal transferase buffer, terminal transferase at a concentration of 20 U/μL, RNase inhibitor at a concentration of 40 U/μL, a total volume of 20 μL, 20 U/μL of terminal transferase and 40 U/μL of RNase inhibitor, terminal transferase The volume ratio of enzyme buffer solution and viral denatured RNA solution is 1:1.3:2.7:8.3, mix well, incubate at 37°C for 60 minutes, incubate at 70°C for 10 minutes to inactivate terminal transferase, and terminate the reaction, that is, the viral RNA molecule At least 10 to 50 oligo-A deoxynucleotides were connected to the 3' end of the virus RNA to obtain a tailed viral RNA solution.

3. Purification of tailed viral RNA

Add 30 µL of diethylpyrocarbonate-treated water to the centrifuge tube containing the tailed viral RNA solution. Add in sequence a sodium acetate solution with a concentration of 3mol/L and a pH of 5.2, a nucleic acid co-precipitation agent with a concentration of 5mg/mL, absolute ethanol, a 5mg/mL nucleic acid co-precipitation agent and a 3mol/L sodium acetate solution with a pH of 5.2, Tailed viral RNA solution, diethyl pyrocarbonate-treated water, and absolute ethanol in a volume ratio of 1:1.25:5:7.5:25, mixed well, centrifuged at 12,000 rpm at 4°C for 35 minutes, and pre-cooled at 4°C Wash the salt with 75% ethanol, centrifuge at 12,000 rpm at 4°C for 5 minutes, discard the supernatant, wait until the ethanol has evaporated, add 20 μL of diethyl pyrocarbonate to treat the water to dissolve the tailed virus RNA, and obtain the purified tailed virus RNA solution .

4. Reverse transcription reaction of tailed viral RNA

Add the purified tailed viral RNA solution to the centrifuge tube at a concentration of Oligo dT primer or modified Oligo dT primer, diethyl pyrocarbonate treated water to 10 μL, Oligo dT primer or modified Oligo dT primer and the purified adducted The volume ratio of tailed virus RNA solution and diethyl pyrocarbonate-treated water is 1:3:6, mix well, incubate the centrifuge tube at 70°C for 10 minutes to denature the purified tailed virus RNA solution, and place on ice Cool on top for 2 to 10 minutes, add a strand buffer, dithiothreitol at a concentration of 0.1mol/L, and deoxyribonucleoside triphosphate at a concentration of 10mmol/L into the centrifuge tube containing the denatured tailed viral RNA solution , reverse transcriptase at a concentration of 200U/μL, RNase inhibitor at a concentration of 40U/μL, 200U/μL of reverse transcriptase and 40U/μL of RNase inhibitor, 0.1mol/L of dithiothreitol, The volume ratio of 10mmol/L deoxyribonucleoside triphosphate, one-strand buffer, and denatured tailed viral RNA solution is 1:1:2:2:4:10, and after mixing, incubate at 50°C for 60 minutes. Incubate at 70°C for 15 minutes to inactivate the reverse transcriptase, terminate the reaction, and obtain viral reverse transcription products.

5. Polymerase chain reaction amplification of viral reverse transcription products

Add viral reverse transcription product, DNA polymerase buffer, deoxyadenosine triphosphate at a concentration of 10mmol/L, upstream primer at a concentration of 10mmol/L, Oligo dT primer or UAP primer at a concentration of 10mmol/L, and a concentration of 5U/μL TaqDNA polymerase, sterile water, total volume 20μL, UAP primer is consistent with the 5' terminal nucleotide sequence of the modified Oligo dT primer, DNA polymerase buffer and 10mmol/L upstream primer, 10mmol/L The volume ratio of Oligo dT primer or UAP primer, 5U/μL TaqDNA polymerase, 10mmol/L deoxyadenosine triphosphate, reverse transcription product, and sterile water is 1:0.1～0.3:0.1～0.3:0.05～ 0.25: 0.4~1: 0.5~1.5: 5.7~7.9, mix evenly, perform polymerase chain reaction according to the conventional method, and obtain the polymerase chain reaction product.

6. Sequencing and analyzing the sequencing results

The resulting polymerase chain reaction products were sent to a sequencing company for sequencing.

In step 4 of the reverse transcription reaction of the tailed viral RNA of the present invention, add the purified tailed viral RNA solution, the modified Oligo dT primer to the centrifuge tube, the ethyl pyrocarbonate treated water to 10 μL, and the modified Oligo dT primer The optimal volume ratio of the purified tailed viral RNA solution and diethyl pyrocarbonate treated water is 1:3:6, mix well, and incubate the centrifuge tube at 70°C for 10 minutes for the purified tailed viral RNA The solution is denatured, cooled on ice for 2 to 10 minutes, and added to the centrifuge tube containing the denatured tailed viral RNA solution, adding a chain buffer, dithiothreitol at a concentration of 0.1mol/L, and a concentration of 10mmol/L deoxyribonucleoside triphosphate, concentration of 200U/μL reverse transcriptase, concentration of 40U/μL RNase inhibitor, 200U/μL reverse transcriptase and 40U/μL RNase inhibitor, 0.1mol/L The optimal volume ratio of dithiothreitol, 10mmol/L deoxyribonucleoside triphosphate, one-strand buffer, and tailed viral RNA solution after denaturation is 1:1:2:2:4:10, mixed After homogenization, incubate at 50°C for 60 minutes, and incubate at 70°C for 15 minutes to inactivate the reverse transcriptase, terminate the reaction, and obtain the virus reverse transcription product.

In the polymerase chain reaction amplification step 5 of the reverse transcription product of the present invention, in the polymerase chain reaction amplification step 5 of the reverse transcription product, the virus reverse transcription product, DNA polymerase buffer are added to the centrifuge tube , deoxyadenosine triphosphate at a concentration of 10mmol/L, upstream primers at a concentration of 10mmol/L, primers at a concentration of 10mmol/LUAP, Taq DNA polymerase at a concentration of 5U/μL, sterile water, and a total volume of 20μL, DNA polymerase The optimal volume ratio of buffer to 10mmol/L upstream primer, 10mmol/L UAP primer, 5U/μL Taq DNA polymerase, 10mmol/L deoxyadenosine triphosphate, reverse transcription product, and sterile water is 1: 0.2:0.2:0.1:0.5:1:7, mix well, and perform polymerase chain reaction according to the conventional method to obtain the polymerase chain reaction product.

The invention can accurately determine the 3' terminal sequence of the viral RNA molecule by means of the reverse transcription reaction after adding the tail through the terminal transfer reaction, which is convenient and quick. Since Oligo dT or modified Oligo dT primers can be used for reverse transcription reaction, the process of designing and synthesizing specific primers for a specific viral RNA molecule is omitted, which greatly simplifies the experimental cycle and saves experimental costs. At the same time, the present invention can also be applied to the determination of the 3' end sequence of other RNA molecules that do not have a PolyA tail structure at the 3' end.

Description of drawings

Figure 1 is a computer software comparison diagram of the rabies virus polymerase chain reaction product sequence RV and the rabies virus genome sequence gene.

Fig. 2 is a computer software comparison diagram of the influenza virus polymerase chain reaction product sequence NP and the influenza virus nucleoprotein gene sequence.

Fig. 3 is a computer software comparison diagram of the polymerase chain reaction product sequence 28SrRNAα of Schistosoma mansoni and the gene sequence of the 28SrRNAα subunit of Schistosoma mansoni.

Example 1

The method for obtaining the 3' end sequence of rabies virus (Rabies virus) genome is an example, and its steps are as follows:

1. Extraction of rabies virus RNA

Rabies virus (ERA strain) was purchased from China Center for Veterinary Microorganism Culture Collection. After inoculating the hamster kidney cells BHK-21 with the virus, culture them in an incubator at 33-35°C for 5 days. The infected cell culture was collected, and the rabies virus RNA was extracted with the QIAamp Viral RNA Mini Kit kit according to the instruction manual. The QIAamp Viral RNA Mini Kit kit is a commercially available commodity sold by Qiagen. Rabies virus RNA was dissolved in 40 μL diethyl pyrocarbonate-treated water, 1 μL was taken to test the sample concentration, and the concentration of rabies virus RNA was quantified to 1 μg/μL.

2. Terminal transfer reaction of rabies virus RNA

Add 7.5 μL of rabies virus RNA, 1 μL of deoxyadenosine triphosphate with a concentration of 10 mmol/L to the centrifuge tube, add diethyl pyrocarbonate-treated water to 12.5 μL, 10 mmol/L of deoxyadenosine triphosphate and diethyl pyrocarbonate The volume ratio of ester-treated water and rabies virus RNA is 1:4:7.5, mix well, incubate at 70° C. for 15 minutes to denature rabies virus RNA, obtain denatured rabies virus RNA solution, and place it on ice for 2 to 10 minutes. Add 4 μL of terminal transferase buffer, 1.5 μL of terminal transferase at a concentration of 20 U/μL, and 2 μL of RNase inhibitor at a concentration of 40 U/μL to the centrifuge tube containing the denatured rabies virus RNA solution sample, for a total volume of 20 μL, 20 U/μL The volume ratio of terminal transferase to 40U/μL RNase inhibitor, terminal transferase buffer, and virus denatured RNA solution is 1: 1.3: 2.7: 8.3, and the terminal transferase buffer, terminal transferase, RNase inhibitor It is a commodity sold on the market, sold by Fermantas, mixed well, incubated at 37°C for 60 minutes, incubated at 70°C for 10 minutes to inactivate terminal transferase, terminated the reaction, and then ligated 10 to the 3' end of the viral RNA molecule. ~50 oligo-A deoxynucleotides to obtain a tailed rabies virus RNA solution.

3. Purification of tailed rabies virus RNA

Add 30 µL of diethylpyrocarbonate-treated water to the centrifuge tube containing 20 µL of tailed rabies virus RNA solution. Add 5 μL of sodium acetate solution with a concentration of 3 mol/L and a pH of 5.2, 4 μL of a nucleic acid co-precipitation agent with a concentration of 5 mg/mL, 100 μL of absolute ethanol, 5 mg/mL of a nucleic acid co-precipitation agent and 3 mol/L sodium acetate solution with a pH of 5.2 , tailed rabies virus RNA solution, diethyl pyrocarbonate treated water, and absolute ethanol have a volume ratio of 1: 1.25: 5: 7.5: 25, and the nucleic acid co-precipitation agent is a commodity sold on the market, produced by Dalian Bao Biological Engineering Co., Ltd. Company sales, mix well, centrifuge at 12,000 rpm at 4°C for 35 minutes, wash the salt with 75% ethanol pre-cooled at 4°C, centrifuge at 12,000 rpm at 4°C for 5 minutes, discard the supernatant, wait until the ethanol has evaporated, add Dissolve the tailed rabies virus RNA in 20 μL of diethyl pyrocarbonate-treated water to obtain a purified tailed virus RNA solution.

4. Reverse transcription of tailed rabies virus RNA

Add 3 μL of purified tailed rabies virus RNA solution to the centrifuge tube, 1 μL concentration is 10 mmol/LOligodT-NUP primer, Oligo dT-NUP primer sequence is ctgatctaga ggtaccggat cctttttttt tttttttttt vn was synthesized by Nanjing GenScript Biotechnology Co., Ltd., 6 μL Diethyl pyrocarbonate-treated water to 10 μL, the volume ratio of 10mmol/L Oligo dT-NUP primer to the purified tailed virus RNA solution and diethyl pyrocarbonate-treated water was 1:3:6, mixed well, and Incubate the centrifuge tube at 70°C for 10 minutes to denature the purified tailed rabies virus RNA solution, place it on ice for 2 to 10 minutes, and add 4 μL of a strand to the centrifuge tube containing the denatured tailed rabies virus RNA solution. Buffer, 2 μL of dithiothreitol at a concentration of 0.1mol/L, 2 μL of deoxyribonucleoside triphosphate at a concentration of 10 mmol/L, 1 μL of reverse transcriptase at a concentration of 200 U/μL, and 1 μL of 40 U/μL RNA Enzyme inhibitors, 200U/μL reverse transcriptase and 40U/μL RNase inhibitor, 0.1mol/L dithiothreitol, 10mmol/L deoxyribonucleoside triphosphate, one-strand buffer, after denaturation The volume ratio of the tailed virus RNA solution is 1: 1: 2: 2: 4: 10, a chain buffer, dithiothreitol, and reverse transcriptase are sold by Invitrogen, USA, and deoxyribonucleoside triphosphate is sold by Roche The RNase inhibitor is sold by the company, and the RNase inhibitor is sold by the Fermantas company. After mixing, incubate at 50°C for 60 minutes, and incubate at 70°C for 15 minutes to inactivate the reverse transcriptase, terminate the reaction, and obtain the tailed rabies virus RNA reverse transcription product.

5. Polymerase chain reaction amplification of tailed rabies virus RNA reverse transcription product

Rabies virus RNA reverse transcripts were amplified by nested polymerase chain reaction. Add 1 μL tailed rabies virus RNA reverse transcription product to the centrifuge tube, 2 μL ExTaq DNA polymerase buffer, 0.8 μL deoxyribonucleoside triphosphate with a concentration of 10 mmol/L, 0.2 μL RV-R ₁ with a concentration of 10 mmol/L Primer, 0.2 μL UAP primer with a concentration of 10mmol/L, 0.1 μL ExTaq DNA polymerase with a concentration of 5U/μL, 15.7 μL sterile water, mix well, ExTaq DNA polymerase buffer and 10mmol/L UAP primer, 10mmol /L RV-R ₁ primer, 5U/μL ExTaq DNA polymerase, 10mmol/L deoxyadenosine triphosphate, reverse transcription product, sterile water volume ratio is 1:0.1:0.1:0.05:0.4:0.5: 7.9, the sequence of the UAP primer is ctgatctaga ggtaccggat cc, the sequence of the RV-R ₁ primer is ggagacttcc cactcaagc, the UAP primer and the RV-R ₁ primer are synthesized by Nanjing KingScript Biotechnology Co., Ltd., mix well and carry out 35 times of polymerase chain Reaction amplification reaction cycle, each cycle is denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 1 minute to obtain the product of the first round of nested polymerase chain reaction of rabies virus; Dilute the polymerase chain reaction product 50 times with sterile water to obtain the diluted product of the first round of nested polymerase chain reaction, add 1 μL of the diluted product of the first round of nested polymerase chain reaction to the centrifuge tube, and polymerize with 2 μL of ExTaq DNA Enzyme buffer, 0.8 μL concentration of 10 mmol/L deoxyribonucleoside triphosphate, 0.2 μL concentration of 10 mmol/L RV-R ₂ primer, 0.2 μL concentration of 10 mmol/L UAP primer, 0.1 μL concentration of 5 U/μLE ExTaq DNA polymerization Enzyme, 15.7 μL sterile water, ExTaq DNA polymerase buffer, 10 mmol/L UAP primer, 10 mmol/L RV-R ₂ primer, 5 U/μL ExTaq DNA polymerase, 10 mmol/L deoxyadenosine triphosphate, The first round of nested polymerase chain reaction dilution product, the volume ratio of sterile water is 1:0.1:0.1:0.05:0.4:0.5:7.9, the sequence of RV- _R2 primer is aacccagtaa acgacaccag, RV- _R2 primer It is synthesized by Nanjing KingScript Biotechnology Co., Ltd. After mixing, it performs 35 cycles of polymerase chain reaction amplification reaction. Each cycle is denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 50 seconds. The product of the second round of nested polymerase chain reaction of rabies virus was obtained; ExTaq DNA polymerase buffer and ExTaq DNA polymerase were products sold on the market, sold by Dalian Bao Biological Engineering Co., Ltd., and deoxyribonucleoside triphosphate was marketed upper pin sold by Roche Corporation.

6. Sequencing and analysis of sequencing results

The product of the second round of nested polymerase chain reaction of rabies virus was sent to Nanjing KingScript Biotechnology Co., Ltd. for sequencing, and the sequence of the polymerase chain reaction product was obtained, which was named RV. For the specific sequence, see the nucleotide sequence table RV.

This sequence is compared with the rabies virus genome sequence (GenBank: EF206707) reported in GenBank with online analysis software (http://www.ebi.ac.uk/Tools/msa/clustalw2/), see Fig. 1 in Fig. In 1, * indicates that the two sequences are completely identical nucleotides, the underline indicates the sequence of the upstream primer RV-R ₂ used, and the rest are non-identical nucleotides. It can be seen from Figure 1 that the 3' ends of the two sequences are completely consistent, indicating that the method can accurately determine the 3' end sequences of RNA molecules.

Example 2

Take the method for positioning the 3' end sequence of the influenza virus nucleoprotein (Nucleoprotein, NP) gene as an example, and its steps are as follows:

1. Extraction of influenza virus RNA

Influenza virus (A/Hubei/1190/2010 (H3N2)) was purchased from China Center for Type Culture Collection. After inoculating the chicken embryos with the virus, culture the chicken embryos in a 33-35°C incubator for 2-3 days. Chicken embryo allantoic fluid was collected, and viral RNA was extracted with the QIAamp Viral RNA Mini Kit kit according to the instructions. The QIAamp Viral RNA Mini Kit kit is commercially available and sold by Qiagen. Viral RNA was dissolved in 40 μL diethylpyrocarbonate-treated water, 1 μL was taken to test the sample concentration, and the concentration of influenza virus RNA was quantified to 1 μg/μL.

2. Terminal transfer reaction of influenza virus RNA

Add 7.5 μg of influenza virus RNA, 1 μL of deoxyadenosine triphosphate at a concentration of 10 mmol/L to the centrifuge tube, add diethyl pyrocarbonate-treated water to 12.5 μL, 10 mmol/L of deoxyadenosine triphosphate and diethyl pyrocarbonate The volume ratio of ester-treated water to influenza virus RNA was 1:4:7.5, mixed evenly, and incubated at 70°C for 15 minutes to denature influenza virus RNA to obtain denatured influenza virus RNA solution, which was immediately placed on ice for 2 to 10 minutes, and poured into Add 4 μL of terminal transferase buffer, 1.5 μL of terminal transferase with a concentration of 20 U/μL, 2 μL of RNase inhibitor with a concentration of 40 U/μL, and 20 U/μL of terminal transferase with 40 U The volume ratio of RNase inhibitor, terminal transferase buffer, and denatured influenza virus RNA per μL is 1:1.3:2.7:8.3. After mixing, incubate at 37°C for 60 minutes, and incubate at 70°C for 10 minutes to make terminal transferase After inactivation and termination of the reaction, 10 to 50 oligo-A deoxynucleotides can be connected to the 3' end of the influenza virus RNA molecule to obtain a tailed influenza virus RNA solution.

3. Purification of tailed influenza virus RNA

Add 30 µL of diethylpyrocarbonate-treated water to the centrifuge tube containing 20 µL of tailed viral RNA. Add 5 μL of sodium acetate solution with a concentration of 3 mol/L and a pH of 5.2, 4 μL of a nucleic acid co-precipitation agent with a concentration of 5 mg/mL, 100 μL of absolute ethanol, 5 mg/mL of a nucleic acid co-precipitation agent and 3 mol/L sodium acetate with a pH of 5.2 solution, tailed influenza virus RNA solution, diethyl pyrocarbonate-treated water, and absolute ethanol at a volume ratio of 1:1.25:5:7.5:25, mix well, and centrifuge at 12,000 rpm at 4°C for 35 minutes. Wash the salt with pre-cooled 75% ethanol, centrifuge at 12,000 rpm at 4°C for 5 minutes, discard the supernatant, wait until the ethanol has evaporated, add 20 μL of diethyl pyrocarbonate to treat the water to dissolve the tailed viral RNA, and obtain the purified Add tailed influenza virus RNA solution.

4. Reverse transcription reaction of tailed influenza virus RNA

Add 3 μL of purified tailed influenza virus RNA, 1 μL of Oligo dT-NUP primer at a concentration of 10 mmol/L, 6 μL of diethylpyrocarbonate-treated water to 10 μL, and 10 mmol/L of Oligo dT-NUP primer and purified The volume ratio of the tailed influenza virus RNA solution and diethyl pyrocarbonate treated water was 1:3:6, mixed evenly, and the centrifuge tube was incubated at 70° C. for 10 minutes to carry out the purification of the tailed influenza virus RNA solution. Denature, place on ice for 2 to 10 minutes, add 4 μL of first-strand buffer, 2 μL of 0.1 mol/L dithiothreitol, 2 μL of 10 mmol/L dithiothreitol into the centrifuge tube containing denatured tailed influenza virus RNA solution /L deoxyribonucleoside triphosphate, 1 μL concentration of 200U/μL reverse transcriptase, 1 μL concentration of 40U/μL RNase inhibitor, 200U/μL reverse transcriptase and 40U/μL RNase inhibitor, The volume ratio of the dithiothreitol of 0.1mol/L, the deoxyribonucleoside triphosphate of 10mmol/L, the one-strand buffer solution, the tailed influenza virus RNA solution after denaturation is 1:1:2:2:4: 10. After mixing, incubate at 50°C for 60 minutes, incubate at 70°C for 15 minutes to inactivate the reverse transcriptase, terminate the reaction, and obtain the reverse transcription product of influenza virus RNA.

5. Polymerase chain reaction amplification of influenza virus RNA reverse transcription product

Add 2 μL tailed viral RNA reverse transcription product, 2 μL ExTaq DNA polymerase buffer, 1 μL deoxyribonucleoside triphosphate with a concentration of 10 mmol/L, 0.4 μL Bm-NP-1565R primer with a concentration of 10 mmol/L, 0.4μL 10mmol/L UAP primer, 0.2μL ExTaq DNA polymerase at a concentration of 5U/μL, 14μL sterile water, ExTaq DNA polymerase buffer, 10mmol/L UAP primer, 10mmol/L Bm-NP-1565R primer, 5U/L The volume ratio of μL of ExTaq DNA polymerase, 10mmol/L of deoxyadenosine triphosphate, and reverse transcription product is 1:0.2:0.2:0.1:0.5:1:7, and the sequence of Bm-NP-1565R primer is atatcgtctc gtattagtag aaacaagggt attttt, Bm-NP-1565R primers were synthesized by Nanjing KingScript Biotechnology Co., Ltd., mixed well and performed 35 cycles of PCR amplification, each cycle was denaturation at 94°C for 30 seconds and annealing at 63°C for 30 seconds Polymerize at 72°C for 2 minutes to obtain a polymerase chain reaction product.

6. Sequencing and analyzing the sequencing results

The influenza virus polymerase chain reaction product was sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing, and the sequence of the polymerase chain reaction product was obtained, which was named NP. See the sequence listing for the specific sequence.

The sequence was compared with the influenza virus nucleoprotein (Nucleoprotein, NP) gene sequence (GenBank: AF255753) reported in GenBank with online analysis software (http://www.ebi.ac.uk/Tools/msa/clustalw2/ ), see Fig. 2, in Fig. 2, * represents two identical nucleotides, the underline represents the sequence of the upstream primer Bm-NP-1565R used, and the rest are non-identical nucleotides. As can be seen from Figure 2, the 3' ends of the two sequences are completely consistent, indicating that the method can accurately determine the 3' end sequence of the RNA molecule.

Example 3

The method for obtaining the 3' end sequence of the 28S rRNAα subunit of Schistosoma mansoni is an example, and the steps are as follows:

1. Extraction of Schistosoma mansoni RNA

The RNA of Schistosoma mansoni was extracted by one-step method with Tizol reagent, and the quality of the obtained Schistosoma mansoni RNA was detected by formaldehyde denaturing gel electrophoresis. The Tizol reagent was a commercial product sold by Dalian Bao Biological Engineering Co., Ltd. Quantify the concentration of viral RNA to 1 µg/µL.

2. Terminal transfer reaction of Schistosoma mansoni RNA

Add 7.5 μg of Schistosoma mansoni RNA, 1 μL of 10 mmol/L deoxyadenosine triphosphate, diethyl pyrocarbonate-treated water to 12.5 μL, and 10 mmol/L of deoxyadenosine triphosphate and diethyl pyrocarbonate in order to the centrifuge tube The volume ratio of ester-treated water and Schistosoma mansoni RNA is 1:4:7.5, mix well, incubate at 70°C for 15 minutes to denature the RNA of Schistosoma mansoni, obtain denatured Schistosoma mansoni RNA solution, and place it on ice for 2-10 minutes , add 4 μL of terminal transferase buffer, 1.5 μL of terminal transferase at a concentration of 20 U/μL, 2 μL of RNase inhibitor at a concentration of 40 U/μL, and 20 U/μL of terminal transferase into the centrifuge tube containing denatured Schistosoma mansoni RNA solution. The volume ratio of the enzyme to 40U/μL of RNase inhibitor, terminal transferase buffer, and denatured Schistosoma mansoni RNA solution is 1:1.3:2.7:8.3. After mixing, incubate at 37°C for 60 minutes, and incubate at 70°C for 10 minutes. The terminal transferase is inactivated within 1 minute, and the reaction is terminated, that is, 10 to 50 oligo-A deoxynucleotides are connected to the 3' end of the Schistosoma mansoni RNA molecule to obtain a Schistosoma mansoni RNA solution.

3. Purification of tailed RNA from Schistosoma mansoni

Add 30 µL of diethylpyrocarbonate-treated water to the centrifuge tube containing 20 µL of S. mansoni RNA solution. Add 5 μL of 3 mol/L sodium acetate solution at pH 5.2, 4 μL of nucleic acid co-precipitation agent with a concentration of 5 mg/mL, 100 μL of absolute ethanol, 5 mg/mL of nucleic acid co-precipitation agent and 3 mol/L sodium acetate solution at pH 5.2 , Schistosoma mansoni RNA solution, diethyl pyrocarbonate-treated water, and absolute ethanol in a volume ratio of 1: 1.25: 5: 7.5: 25, mix well, centrifuge at 12,000 rpm at 4°C for 35 minutes, and use 4°C Wash the salt with pre-cooled 75% ethanol, centrifuge at 12,000 rpm at 4°C for 5 minutes, discard the supernatant, wait until the ethanol has evaporated, add 20 μL of diethyl pyrocarbonate to treat the water to dissolve Schistosoma mansoni RNA, and obtain purified Schistosoma mansoni RNA solution.

4. Reverse transcription reaction of Schistosoma mansoni tailed RNA

Add 3 μL of purified Schistosoma mansoni RNA to the centrifuge tube, 1 μL of Oligo dT-NUP primer with a concentration of 10 mmol/L, 6 μL of diethyl pyrocarbonate-treated water to 10 μL, and 10 mmol/L of Oligo dT-NUP primer with The volume ratio of the purified Schistosoma mansoni RNA solution and diethyl pyrocarbonate-treated water is 1:3:6, mix well, and incubate the centrifuge tube at 70°C for 10 minutes to treat the purified Schistosoma mansoni Denature the RNA, place it on ice for 2 to 10 minutes, and add 4 μL of first-strand buffer, 2 μL of dithiothreitol with a concentration of 0.1 mol/L, and 2 μL of Deoxyribonucleoside triphosphate at a concentration of 10 mmol/L, 1 μL of reverse transcriptase at a concentration of 200 U/μL, 1 μL of RNase inhibitor at a concentration of 40 U/μL, 200 U/μL of reverse transcriptase and 40 U/μL of RNase The volume ratio of the dithiothreitol of inhibitor, 0.1mol/L, the deoxyribonucleoside triphosphate of 10mmol/L, a chain buffer, the denatured Schistosoma mansoni RNA solution is 1:1:2: 2:4:10, after mixing, incubate at 50°C for 60 minutes, incubate at 70°C for 15 minutes to inactivate the reverse transcriptase, terminate the reaction, and obtain the reverse transcription product of Schistosoma mansoni RNA.

5. Polymerase chain reaction amplification of Schistosoma mansoni RNA reverse transcription product

Amplification of Schistosoma mansoni RNA reverse transcription products by nested polymerase chain reaction. Add 3 μL of Schistosoma mansoni RNA reverse transcription product, 2 μL of EasyTaq DNA polymerase buffer, 2 μL of deoxyribonucleoside triphosphate at a concentration of 10 mmol/L, and 0.6 μL of 28SαF ₁ primer aagcagaact ggcgctgtgg gatg at a concentration of 10 mmol/L to the centrifuge tube , 0.6μL UAP primer with a concentration of 10mmol/L, 0.5μL EasyTaq DNA polymerase with a concentration of 5U/μL, 11.3μL sterile water, mix well, EasyTaq DNA polymerase buffer and 10mmol/L UAP primer, 10mmol/L L of 28SαF ₁ primer, 5U/μL of EasyTaq DNA polymerase, 10mmol/L of deoxyadenosine triphosphate, the volume ratio of Schistosoma mansoni RNA reverse transcription product is 1:0.3:0.3:0.25:1:1.5:5.7, 28SαF The sequence of ₁ primer is aagcagaact ggcgctgtgg gat, 28SαF ₁ primer was synthesized by Nanjing GenScript Biotechnology Co., Ltd., after mixing, 35 PCR amplification cycles were performed, and each cycle was denatured at 94°C for 30 seconds, 58 Annealing at ℃ for 30 seconds and polymerizing at 72℃ for 50 seconds to obtain the first round of nested polymerase chain reaction product of Schistosoma mansoni; the first round of nested polymerase chain reaction product of Schistosoma mansoni was diluted 50 times with sterile water, To obtain the diluted product of the first round of nested polymerase chain reaction of Schistosoma mansoni, add 3 μL of the diluted product of the first round of nested polymerase chain reaction of Schistosoma mansoni to the centrifuge tube, 2 μL of EasyTaq DNA polymerase buffer, and 2 μL of 10 mmol /L of deoxyribonucleoside triphosphate, 0.6 μL of 10 mmol/L 28SαF ₂ primer, 0.6 μL of 10 mmol/L UAP primer, 0.5 μL of 5 U/μL EasyTaq DNA polymerase, 11.3 μL of sterile water , EasyTaq DNA polymerase buffer with 10mmol/L UAP primer, 10mmol/L 28SαF ₂ primer, 5U/μL EasyTaq DNA polymerase, 10mmol/L deoxyadenosine triphosphate, the first round of nested polymerization of Schistosoma mansoni The volume ratio of the enzyme chain reaction diluted product was 1:0.3:0.3:0.25:1:1.5:5.7, the sequence of the 28SαF ₂ primer was agacakcagg acggtggcca tgg, and the 28SαF ₂ primer was synthesized by Nanjing GenScript Biotechnology Co., Ltd. After homogenization, 35 PCR amplification cycles were performed, each cycle was denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and polymerization at 72°C for 50 seconds to obtain the second round of nested PCR of Schistosoma mansoni product; EasyTaq DNA polymerase buffer, EasyTaq DNA polymerase are commercially available products Products, sold by Beijing Quanshijin Biotechnology Co., Ltd.

6. Sequencing and analyzing the sequencing results

The second-round nested polymerase chain reaction product of Schistosoma mansoni was sent to Nanjing KingScript Biotechnology Co., Ltd. for sequencing, and the sequence of the polymerase chain reaction product was obtained, which was named 28SrRNAα. See the sequence table for the specific sequence.

The partial gene sequence of Schistosoma mansoni 28SrRNA reported in GenBank (GenBank: X13836) is 568bp in length, according to previous research results (van Keulen H, Mertz PM, LoVerde PT, Shi H, Rekosh DM, 1991.Characterization of a 54 -nucleotide gap region in the 28S rRNA gene of Schistosoma mansoni.MolBiochem Parasitol.45(2):205-214.), the 3' end sequence of the 28S rRNAα subunit is at the 138th base. Use this partial sequence to compare with the 28SrRNAα sequence obtained in this example with online analysis software (http://www.ebi.ac.uk/Tools/msa/clustalw2/), see Figure 3, in Figure 3, * indicates the nucleotides that are completely identical in the two sequences, the underline indicates the sequence of the primer 28SαF ₂ used, and the rest are non-identical nucleotides. It can be seen from Figure 3 that the 3' ends of the two sequences are completely consistent, indicating that the method can accurately determine the 3' end sequences of RNA molecules.

Example 4

In the reverse transcription reaction step 4 of the tailed viral RNA in the above embodiments 1-3, the modified Oligo dT primers used were replaced with Oligo dT primers of equal quality, and other steps in this step were the same as the corresponding examples. In the polymerase chain reaction amplification step 5 of the reverse transcription product, the modified Oligo dT primers used were replaced with equal-quality Oligo dT primers, and other steps in this step were the same as in the corresponding examples. Other steps are the same as the corresponding embodiment.

Claims (3)

1. A method for determining the 3' end sequence of the viral RNA molecule, comprising the steps of: (1) Extraction of viral RNA Extract viral RNA by conventional methods, detect the quality of viral RNA obtained, and quantify the concentration of viral RNA to 1 μg/μL; (2) Terminal transfer reaction of viral RNA Add viral RNA, deoxyadenosine triphosphate and diethyl pyrocarbonate-treated water at a concentration of 10mmol/L to 12.5μL, and 10mmol/L deoxyadenosine triphosphate and diethylpyrocarbonate-treated water, virus The volume ratio of the RNA is 1:4:7.5, mix well, incubate at 70°C for 15 minutes to denature the viral RNA, obtain the denatured viral RNA solution, put it on ice for 2-10 minutes, and add it to the centrifuge tube containing the denatured viral RNA solution Terminal transferase buffer, terminal transferase at a concentration of 20 U/μL, RNase inhibitor at a concentration of 40 U/μL, a total volume of 20 μL, 20 U/μL of terminal transferase and 40 U/μL of RNase inhibitor, terminal transferase The volume ratio of enzyme buffer solution and viral denatured RNA solution is 1:1.3:2.7:8.3, mix well, incubate at 37°C for 60 minutes, incubate at 70°C for 10 minutes to inactivate terminal transferase, and terminate the reaction, that is, the viral RNA molecule At least 10 to 50 oligo-A deoxynucleotides are connected to the 3' end of the method to obtain a tailed viral RNA solution; (3) Purification of tailed viral RNA Add 30 µL of diethylpyrocarbonate-treated water to the centrifuge tube containing the tailed viral RNA solution. Add in sequence a sodium acetate solution with a concentration of 3mol/L and a pH of 5.2, a nucleic acid co-precipitation agent with a concentration of 5mg/mL, absolute ethanol, a 5mg/mL nucleic acid co-precipitation agent and a 3mol/L sodium acetate solution with a pH of 5.2, Tailed viral RNA solution, diethyl pyrocarbonate-treated water, and absolute ethanol in a volume ratio of 1:1.25:5:7.5:25, mixed well, centrifuged at 12,000 rpm at 4°C for 35 minutes, and pre-cooled at 4°C Wash the salt with 75% ethanol, centrifuge at 12,000 rpm at 4°C for 5 minutes, discard the supernatant, wait until the ethanol has evaporated, add 20 μL of diethyl pyrocarbonate to treat the water to dissolve the tailed virus RNA, and obtain the purified tailed virus RNA solution ; (4) Reverse transcription reaction of tailed viral RNA Add the purified tailed viral RNA solution to the centrifuge tube at a concentration of Oligo dT primer or modified Oligo dT primer, diethyl pyrocarbonate treated water to 10 μL, Oligo dT primer or modified Oligo dT primer and the purified adducted The volume ratio of tailed virus RNA solution and diethyl pyrocarbonate-treated water is 1:3:6, mix well, incubate the centrifuge tube at 70°C for 10 minutes to denature the purified tailed virus RNA solution, and place on ice Cool on top for 2 to 10 minutes, add a strand buffer, dithiothreitol at a concentration of 0.1mol/L, and deoxyribonucleoside triphosphate at a concentration of 10mmol/L into the centrifuge tube containing the denatured tailed viral RNA solution , reverse transcriptase at a concentration of 200U/μL, RNase inhibitor at a concentration of 40U/μL, 200U/μL of reverse transcriptase and 40U/μL of RNase inhibitor, 0.1mol/L of dithiothreitol, The volume ratio of 10mmol/L deoxyribonucleoside triphosphate, one-strand buffer, and denatured tailed viral RNA solution is 1:1:2:2:4:10, and after mixing, incubate at 50°C for 60 minutes. Incubate at 70°C for 15 minutes to inactivate reverse transcriptase, terminate the reaction, and obtain viral reverse transcription products; (5) Polymerase chain reaction amplification of viral reverse transcription product Add viral reverse transcription product, DNA polymerase buffer, deoxyadenosine triphosphate at a concentration of 10mmol/L, upstream primer at a concentration of 10mmol/L, Oligo dT primer or UAP primer at a concentration of 10mmol/L, and a concentration of 5U/μL TaqDNA polymerase, sterile water, total volume 20μL, UAP primer is consistent with the 5' terminal nucleotide sequence of the modified Oligo dT primer, DNA polymerase buffer and 10mmol/L upstream primer, 10mmol/L The volume ratio of Oligo dT primer or UAP primer, 5U/μL TaqDNA polymerase, 10mmol/L deoxyadenosine triphosphate, reverse transcription product, and sterile water is 1:0.1～0.3:0.1～0.3:0.05～ 0.25: 0.4~1: 0.5~1.5: 5.7~7.9, mix evenly, perform polymerase chain reaction according to the conventional method, and obtain the polymerase chain reaction product. 2. the method for determining viral RNA molecule 3' end sequence according to claim 1 is characterized in that: in the reverse transcription reaction step (4) of adding tail virus RNA, add the tail virus after purification in centrifuge tube RNA solution, modified Oligo dT primer, diethylpyrocarbonate-treated water to 10 μL, the volume ratio of modified Oligo dT primer to purified tailed virus RNA solution, diethylpyrocarbonate-treated water is 1:3:6 , mix well, incubate the centrifuge tube at 70°C for 10 minutes to denature the purified tailed viral RNA solution, cool it on ice for 2-10 minutes, add a Chain buffer, dithiothreitol at a concentration of 0.1mol/L, deoxyribonucleoside triphosphate at a concentration of 10mmol/L, reverse transcriptase at a concentration of 200U/μL, and RNase inhibitor at a concentration of 40U/μL , 200U/μL reverse transcriptase and 40U/μL RNase inhibitor, 0.1mol/L dithiothreitol, 10mmol/L deoxyribonucleoside triphosphate, one-strand buffer, denatured tailing The volume ratio of the viral RNA solution is 1:1:2:2:4:10. After mixing, incubate at 50°C for 60 minutes, and incubate at 70°C for 15 minutes to inactivate the reverse transcriptase, terminate the reaction, and obtain the viral reverse transcription product . 3. The method for determining the 3' end sequence of the viral RNA molecule according to claim 1 or 2, characterized in that: in the polymerase chain reaction amplification step (5) of the reverse transcription product, the virus is added to the centrifuge tube Reverse transcription product, DNA polymerase buffer, deoxyadenosine triphosphate at a concentration of 10mmol/L, upstream primer at a concentration of 10mmol/L, UAP primer at a concentration of 10mmol/L, Taq DNA polymerase at a concentration of 5U/μL, no Bacterial water, total volume 20μL, DNA polymerase buffer, 10mmol/L upstream primer, 10mmol/L UAP primer, 5U/μL Taq DNA polymerase, 10mmol/L deoxyadenosine triphosphate, reverse transcription product, no The bacteria-to-water volume ratio is 1:0.2:0.2:0.1:0.5:1:7, mixed evenly, and polymerase chain reaction is carried out according to a conventional method to obtain a polymerase chain reaction product.

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