TaqMan multiple RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) detection method and kit for four hematogenous diseases

Abstract

The invention discloses a TaqMan multiple RT-qPCR (reverse transcription-quantitative polymerase chain reaction) detection method and kit for four hematogenous diseases, and the method comprises the following steps: extracting virus RNA (Ribonucleic Acid) in a sample: carrying out RNA extraction on four strains of HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), HCV (Hepatitis C Virus) and HDV (Hepatitis D Virus) by using TIANAmp Virus RNAKit to obtain a virus RNA sample; designing a specific primer and a probe: designing the specific primer and the probe according to four strains of HIV, HBV, HCV and HDV; preparing a standard template and optimizing a reaction system and reaction conditions; constructing a standard curve; the sensitivity, the specificity and the repeatability of the TaqMan multiple RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) Sample detection: after the accuracy of the TaqMan multiple RT-qPCR detection method for the four hematogenous diseases is verified, TaqMan multiple RT-qPCR detection can be carried out on four virus samples, and a detection result is judged according to detection data; the invention further discloses application of the TaqMan multiple RT-qPCR detection kit for the four hematogenous diseases to detection of one or more of HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus) and HDV (high density virus).

Description

Technical Field

[0001] The patent of this invention belongs to the field of disease detection technology, and specifically relates to TaqMan multiplex RT-qPCR detection methods and kits for four blood-borne diseases. Background Art

[0002] Human Immunodeficiency Virus (HIV) belongs to the family of retroviruses. When a person is infected with HIV, the virus first attacks immune cells, causing the body's immune capacity to decline, which can easily lead to infection with other pathogens, causing patients to have secondary infections of multiple pathogens, multiple complications, aggravated conditions, and increased mortality and the incidence of malignant tumors.

[0003] Hepatitis B virus (HBV) was first discovered in 1964. It is a DNA virus belonging to the family Hepadnaviridae. HBV is malignant and highly contagious, causing great damage to the liver. If the disease is not controlled, it will cause hepatic cell necrosis, seriously affecting liver function and inducing other diseases, such as hepatitis-induced diabetes, fatty liver, and hyperbilirubinemia.

[0004] Hepatitis C virus (HCV) was discovered in 1974. It belongs to the Flaviviridae family and the genus Hepacivirus. It is a positive-strand RNA virus. After acute HCV infection, about 50%-80% of HCV patients will develop chronic infection, leading to chronic hepatitis, liver fibrosis, cirrhosis, and even serious chronic liver diseases such as liver failure and hepatocellular carcinoma.

[0005] Hepatitis D virus (HDV) belongs to the genus Deltavirus and is a defective RNA virus with a genome length of approximately 1.7 kb. It requires the help of another virus (HBV) to reproduce. Inadequate detection of HDV is the main reason for limited epidemiological data worldwide.

[0006] Approximately 10-15% of mortality among HIV-infected patients is due to liver disease. Most liver diseases are caused by chronic viral hepatitis. Considering that the pathogens that cause viral hepatitis, such as hepatitis B virus (HBV) and hepatitis C virus (HCV), have similar transmission routes to HIV, HIV / HBV / HCV / HDV are prone to multiple infections. HIV / HBV / HCV / HDV have similar transmission routes and are often transmitted through blood, body fluids and sexual behavior. After human infection, they cause AIDS, hepatitis B, hepatitis C and hepatitis D. At present, these diseases are very harmful to human health and have become a public health problem that seriously affects people's normal life. Due to the similar transmission routes of these four viruses, two or more pathogens are often co-infected. Compared with single infection, patients with multiple infections have significantly accelerated liver fibrosis, and the risk of death in patients with liver diseases such as cirrhosis and hepatocellular carcinoma is also higher, the difficulty of treatment increases, and the incidence of poor prognosis is also significantly higher.

[0007] Commonly used pathogen detection methods are mostly fluorescent quantitative PCR for a single pathogen, which has low detection efficiency, slow detection speed, high cost, and missed diagnosis, hindering the early treatment and prevention of these diseases. Therefore, it is very necessary to develop a single-tube rapid nucleic acid detection method for these four pathogens at the same time, which is convenient for rapid detection and effective prevention and control of diseases.

[0008] Therefore, this paper proposes four TaqMan multiplex RT-qPCR detection methods and kits for blood-borne diseases. Summary of the invention

[0009] In order to solve the above problems, the present invention provides a TaqMan multiplex RT-qPCR detection method and kit for four blood-borne diseases. The TaqMan fluorescent quantitative PCR method is adopted to construct a TaqMan multiplex RT-qPCR for four blood-borne diseases, which can simultaneously perform efficient, high-precision and accurate detection of the four blood-borne diseases.

[0010] In order to achieve the above technical effects, the present invention provides four blood-borne disease TaqMan multiplex RT-qPCR detection methods, which specifically include the following steps:

[0011] S1. Extraction of viral RNA from samples: RNA of four strains of HIV, HBV, HCV, and HDV was extracted using TIANamp Virus RNAKit to obtain viral RNA samples;

[0012] S2. Specific primer and probe design: Design specific primers and probes according to HIV, HBV, HCV, and HDV strains;

[0013] Wherein, the sequences of the primers are shown in SEQ ID No.5 to SEQ ID No.12, and the sequences of the probes are shown in SEQ ID No.13 to SEQ ID No.16;

[0014] S3. Preparation of standard templates: Prepare 19T-HBV plasmid standards and HIV, HCV, and HDV in vitro transcribed RNA standards based on viral RNA samples, and use them as standards;

[0015] S4. Optimization of TaqMan multiplex RT-qPCR reaction conditions (only required when establishing the method for the first time): Use specific primer and probe combinations to perform TaqMan multiplex RT-qPCR detection on plasmid standards and RNA standards of HIV, HBV, HCV, and HDV, and optimize the reaction system and reaction conditions;

[0016] Preferably, the reaction system and reaction conditions optimized in S4 are as follows: primers, probes, annealing temperature and reverse transcription temperature of four viruses in a 25 μL reaction system;

[0017] The upstream and downstream primers (10 μmol / L) were used in amounts of 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, and 0.7 μL;

[0018] The probe (10 μmol / L) was used in amounts of 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, and 0.7 μL;

[0019] The annealing temperatures are optimized at 56°C, 58°C, 60°C, and 62°C;

[0020] The reverse transcription temperature is optimized at 48°C, 50°C, 52°C, and 54°C;

[0021] The number of cycles of the TaqMan multiplex RT-qPCR was 40 times.

[0022] S5. Construct a standard curve: Mix equal volumes of the four standards of HIV, HBV, HCV, and HDV, and select 10 9 -10 4 The mixed standard of 1000 copies / μl was used as the template, and ddH2O was used as the negative template. TaqMan multiplex RT-qPCR amplification was performed according to the optimized reaction system and reaction conditions. The amplification efficiency was analyzed by real-time fluorescence quantitative instrument matching software, and the standard curve was drawn according to the linear relationship between the logarithm of the starting copy number of the standard and the CT value of the four viruses.

[0023] S6. Sensitivity, specificity, and repeatability test of TaqMan multiplex RT-qPCR (only required when establishing the method for the first time);

[0024] Preferably, the sensitivity test of multiplex TaqMan fluorescent quantitative PCR in S6 specifically comprises: mixing equal volumes of four standards of HIV, HBV, HCV and HDV, selecting 10 8 ~10 2 The mixed standard of 1000 copy / μl was used as template and ddH2O as negative control. TaqMan multiplex RT-qPCR amplification was performed according to the optimized reaction system and reaction conditions to evaluate the sensitivity of the method.

[0025] Preferably, the specificity test of TaqMan multiplex RT-qPCR in S6 specifically includes: in the HIV, HBV, HCV, HDV TaqMan multiplex RT-qPCR detection method, using HIV, HCV, HDV RNA and HBV plasmid as templates, using common flavivirus viruses dengue virus (DENV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), iridovirus (HT5K), ddH2O as a negative control, and performing TaqMan multiplex RT-qPCR amplification according to the optimized reaction system and reaction conditions to evaluate the specificity of the method.

[0026] Preferably, the repeatability test of TaqMan multiple RT-qPCR in S6 specifically includes: in the HIV, HBV, HCV, HDV TaqMan multiple RT-qPCR detection method, using HIV, HCV, HDV RNA and HBV plasmid as templates, ddH2O as a negative control, according to the optimized reaction system and reaction conditions, selecting 10 9 copy / μl, 10 7 copy / μl, 10 5 The experiment was repeated within and between groups with three gradients of 1:1 copy / μl. The mean value and coefficient of variation (CV) of the obtained CT values ​​were calculated in Excel to evaluate the repeatability of the method.

[0027] S7, sample testing: After the accuracy of the TaqMan multiplex RT-qPCR detection method for four blood-borne diseases has been verified by S6, TaqMan multiplex RT-qPCR detection can be performed on the four virus samples, and the test results can be judged based on the test data;

[0028] The test results are determined by:

[0029] Positive conditions are: CT value of test result <35;

[0030] Negative condition: CT value ≥35.

[0031] Based on the above, the present invention also provides four blood-borne disease TaqMan multiplex RT-qPCR detection kits, the kits include: Takara One Step PrimeScript TM III RT-qPCR Mix (2x), ddH2O, four blood-borne virus mixed primers, four blood-borne virus mixed probes, positive control: four virus standard mixed samples, negative control: ddH2O;

[0032] Among them, the sequences of the four blood-borne virus mixed primers are shown as SEQ ID No.5 to SEQ ID No.12, and the sequences of the four blood-borne virus mixed probes are shown as SEQ ID No.13 to SEQ ID No.16 respectively.

[0033] Preferably, the positive control is a mixed standard containing four blood-borne viruses HIV, HBV, HCV, and HDV, respectively, and the concentration of the positive control is 10 9 ~10 4 copy / μl.

[0034] Based on the above, the present invention also provides the use of four blood-borne disease TaqMan multiplex RT-qPCR detection kits in detecting one or more of HIV, HBV, HCV, and HDV.

[0035] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0036] The present invention has developed TaqMan multiplex RT-qPCR detection methods and reagents for four blood-borne diseases, namely HIV, HBV, HCV, and HDV. The nucleic acids of the four viruses can be quickly and efficiently, with high precision, and accurately detected in a single tube, which is convenient for rapid detection and effective prevention and control of diseases. Moreover, the method can be repeatedly established, which is conducive to promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for describing the embodiment are briefly introduced below. Those skilled in the art can also obtain other drawings based on these drawings without paying any creative work.

[0038] Figure 1 After mixing the four standard products of the present invention in equal volumes, select 10 9 -10 4 Standard curve of the mixed standard with copy / μl as template;

[0039] Figure 2 After mixing the four standard products of the present invention in equal volumes, select 109 -10 4 Amplification curve of the mixed standard of 1000 copies / μl as template;

[0040] Figure 3 It is the amplification curve diagram of the sensitivity test of the present invention;

[0041] Figure 4 It is the amplification curve diagram of the specific control test of the present invention; DETAILED DESCRIPTION

[0042] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] Because the four commonly used virus detection methods for HIV, HBV, HCV, and HDV are mostly fluorescent quantitative PCR targeting a single pathogen, they have low detection efficiency, slow detection speed, high cost, and may lead to missed diagnoses, which hinders the early treatment and prevention of these diseases.

[0045] Therefore, the inventors provided four blood-borne disease TaqMan multiplex RT-qPCR detection methods;

[0046] Among them, experimental materials: the information of four blood-borne virus DNA target sequences is as follows:

[0047]

[0048]

[0049] Specific experimental steps:

[0050] S1. Extraction of viral RNA from samples: RNA of four strains of HIV, HBV, HCV, and HDV was extracted using TIANamp Virus RNAKit to obtain viral RNA samples;

[0051] The extraction steps specifically include:

[0052] 1. Preparation: When using the kit for the first time, add 11.8 ml and 48 ml of anhydrous ethanol to buffer GD and rinse solution RW respectively. Add 310 μl of RNase-Free ddH2O to the EP tube containing 310 μg Carrier RNA powder, fully dissolve, divide according to experimental needs, and store at -20℃.

[0053] 2. Preparation of Carrier RNA working solution: For each sample, take 5.6 μl of Carrier RNA aqueous solution and add it to 560 μl, then invert and mix.

[0054] 3. Prepare a 1.5ml EP tube for each sample, add 140μl of the strain, then add 560μl of the working solution, vortex for 15s to mix, incubate at room temperature for 10min, and centrifuge instantly; then add 560μL of anhydrous ethanol, cover the tube, vortex for 15s, mix thoroughly, and centrifuge instantly;

[0055] 4. Carefully transfer 630 μl of the above mixed solution from the EP tube to the RNase-Free adsorption column CR2, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

[0056] 5. Repeat the previous step and pass the remaining liquid in the EP tube through the column again according to S104; add 500 μl buffer GD, centrifuge at 12000 rpm for one minute, discard the waste liquid, and put the adsorption column back into the collection tube;

[0057] 6. Add 500 μl of rinse solution RW, centrifuge at 12,000 rpm for one minute, discard the waste liquid, put the adsorption column back into the collection tube and repeat this step;

[0058] 7. Drying: Centrifuge at 12000rpm for 3min; then put the adsorption column into a new 1.5mL EP tube, open the cover of the adsorption column, and leave it at room temperature for 3min to allow the adsorption film to completely dry;

[0059] 8. Add 30 μL RNase-Free ddH2O in the middle of the adsorption column, cover the lid and incubate at room temperature for 3 minutes, centrifuge at 12000 rpm for 1 minute, discard the adsorption column, and finally store the extracted RNA in a -80℃ refrigerator.

[0060] S2. Specific primer and probe design: Design specific primers and probes according to HIV, HBV, HCV, and HDV strains;

[0061] Wherein, the sequences of the primers are shown in SEQ ID No.5 to SEQ ID No.12, and the sequences of the probes are shown in SEQ ID No.13 to SEQ ID No.16;

[0062]

[0063] S3. Preparation of standard templates: Prepare 19T-HBV plasmid standards and HIV, HCV, and HDV in vitro transcribed RNA standards based on viral RNA samples, and use them as standards;

[0064] The preparation steps specifically include:

[0065] 1. Preparation of cDNA: Reverse transcribe the extracted RNA. The reaction system and procedure are as follows:

[0066] Reverse transcription reaction system

[0067]

[0068]

[0069] The reaction conditions are as follows: Random Primerpd(N)6, dNTP Mixture (10mM), and RNA are mixed and centrifuged at 65°C for 10 minutes, and then taken out and incubated on ice for 2 minutes; M-MLV Buffer, ReverseTranscriptase M-MLV, ddH2O, and RRI are added to the above mixture, mixed and centrifuged, incubated at 42°C for 60 minutes, and incubated at 70°C for 10 minutes.

[0070] 2. In vitro transcription, preparation of HIV / HBV / HCV plasmids, amplification of HIV / HBV / HCV target genes (HDV was synthesized by Shanghai Shenggong Biotechnology Co., Ltd.): HIV / HCV cDNA was used as a template, HBV DNA was used as a template, and ordinary PCR amplification was performed according to the reaction system and reaction conditions using the designed specific primers;

[0071] Common PCR reaction system

[0072] Reaction components Dosage (μl) 10×ExTaqBuffer 2 dNTPMixture 2 ExTaq 0.3 F 0.5 R 0.5 <![CDATA[ddH2O]]> 12.7 template 2 Total 20

[0073] 3. Agar gel electrophoresis: first prepare 1% agarose gel solution, then prepare gel plates, wash the agar gel plate slot, insert the tooth comb and transparent gel into the plate slot to fix it, pour in the agar gel solution, cool to room temperature until the gel solution solidifies into a gel block; then load the sample on the gel plate, and finally perform electrophoresis;

[0074] 4. Purification of target fragments: Use Biospin gel recovery and PCR product purification kit to cut and recover the electrophoresis product in S4 to obtain purified PCR products. The specific steps include:

[0075] (1) Preparation: Prepare the corresponding number of autoclaved 1.5 mL EP tubes for sample recovery and number them. Before use, add anhydrous ethanol according to the volume indicated on the label of the Wash Buffer bottle and mix them well. Under ultraviolet light, determine the position of the target band, cut the gel with PCR products and place it in the prepared 1.5 mL EP tube. Pay attention to ensuring the integrity and reducing the gel volume as much as possible.

[0076] (2) Add the gel solution Extraction Buffer in a ratio of 1:3 and incubate at 50°C for 10 min until the gel is fully dissolved. Transfer the entire mixture to the spin column, centrifuge at 12,000 rpm for 1 min, and discard the solution.

[0077] (3) Add 500 μl Extraction Buffer to the Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the solution; add 750 μl Wash Buffer to the Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the solution;

[0078] (4) Drying: Centrifuge at 12000 rpm for 1 min. Transfer the Spin Column to a 1.5 ml EP tube; add 30 μl of preheated Elution Buffer to the Spin Column, place at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, discard the Spin Column, collect the filtrate, and store at -20°C.

[0079] 5. Connect the target gene fragments: Connect the products according to the instructions of pMD19-T vector and PUC57 vector. The connection system is shown in the table:

[0080] PMD19-Vector connection system

[0081] Reaction components Dosage (μl) PMD19-Vector 1 solutionI 5 Purification of PCR products 6 Total 12

[0082] Reaction conditions: After mixing and centrifugation, connect in a metal bath at 16°C for 4 hours or overnight.

[0083] PUC57 vector ligation system

[0084] Reaction components Dosage (μl) pUC57-simpleEVL 1 2×SeamlessCloningMix 5 Purification of PCR products 6 Total 12

[0085] Reaction conditions: Mix well and centrifuge, then incubate at 50°C for 15 min on a PCR instrument.

[0086] 6. Transformation: Take out the DH5α competent cells and melt them, prepare the connected DNA and the corresponding number of 1.5mL sterile EP tubes and number them, take 30μL DH5α competent cells, transfer them into the EP tube, add the corresponding DNA and mix well, centrifuge briefly; then place them on ice for 30 minutes, open the water bath at 42℃ for 1 minute, take them out, put them in the ice box for 5-6 minutes; then add 500μL of non-resistant LB liquid culture medium (LB-) to each EP tube, shake at 37℃, 180rpm / min for 1 hour, finally take 100μL of the incubated bacterial solution and evenly spread it on the LB plate containing ampicillin resistance, and culture it in a 37℃ incubator overnight;

[0087] 7. Screening and identification of positive clones: After overnight culture on the culture plate, pick bacteria on the plate. Use a 2.5μl pipette tip that has been sterilized at high temperature to pick 8 single E. coli colonies with neat edges and smooth shapes on each plate and place them in a 1.5mL EP tube. Add 800μl of LB medium (LB+) containing ampicillin resistance, and culture overnight at 37℃ air shaker at 180rmp / min. Use the cultured bacterial solution as a template for ordinary PCR amplification. The amplification system is as follows:

[0088]

[0089] Reaction conditions: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s (HIV 56℃, HBV / HCV 55℃)

[0090] Anneal for 30 s, extend at 72 °C for 1 min, for a total of 35 cycles; 72 °C for 10 min, hold at 4 °C.

[0091] Gel electrophoresis was performed to observe the results, and the PCR products of positive samples were sent to Shanghai Bioengineering Co., Ltd. for sequencing and verification.

[0092] 8. Bacterial expansion: The synthetic HDV bacterial solution and the bacterial solution with positive gel electrophoresis results were expanded. Take 100 μl of the bacterial solution, add 20 ml of LB medium (LB+) containing ampicillin resistance, and culture overnight at 37°C air shaker at 180 rpm to preserve the bacterial solution. Prepare 5 sterile EP tubes for each strain, take 500 μl of the overnight bacterial solution and 80% glycerol, shake and mix, and store at -80°C;

[0093] 9. Plasmid extraction: Use TaKaRa MiniBEST Plasmid Purification Kit Ver. 4.0 to extract plasmids from HIV, HBV, HCV, and HDV to obtain PUC57-HIV plasmid, PUC57-HCV plasmid, PUC57-HDV plasmid, and pMD19T-HBV plasmid. The specific steps include:

[0094] (1) Preparation: When using for the first time, add 56 ml of anhydrous ethanol to Buffer WB. When using for the first time, add all the RNase A turbid solution to Solution I, mix and store at 4°C. Before using Solution III, place it in a 4°C refrigerator to precool.

[0095] (2) Take 2 ml of overnight culture solution and add it to a sterile EP tube. Centrifuge at 12000 rpm for 2 min and discard the supernatant.

[0096] (3) Use 250 μl of Solution I containing RNase A to fully suspend the bacterial precipitate. Be careful not to leave any small bacterial clumps. Use a vortex or other vigorous shaking method to fully suspend the bacterial cells.

[0097] (4) Add 250 μl of Solution II to a sterile EP tube and gently invert for 5 to 6 times to fully lyse the bacteria and form a transparent solution. Do not shake vigorously during this step and the mixture should not exceed 5 minutes.

[0098] (5) Take out Solution III precooled at 4°C from the refrigerator, add 350 μl to a sterile EP tube, gently invert and mix 5 to 6 times until a compact agglomerate is formed, and then let it stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 10 minutes at room temperature and collect the supernatant.

[0099] (6) Place the Spin Column in the kit on the Collection Tube. Transfer the supernatant in the EP tube to the Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0100] (7) Add 500 μl of Buffer WA to the Spin Column, centrifuge at 12,000 rpm for 30 s, and discard the filtrate.

[0101] (8) Add 700 μl of Buffer WB to the Spin Column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0102] (9) Repeat the previous step and spin dry: Place the Spin Column back on the Collection Tube and centrifuge at 12,000 rpm for 1 minute to remove all residual wash solution.

[0103] (10) Place the Spin Column in a new 1.5 ml sterile EP tube, add 30 μl of preheated Elution Buffer to the center of the Spin Column membrane, and let stand at room temperature for 1 minute.

[0104] (11) Centrifuge at 12000 rpm for 1 min to elute the DNA, label it, and store it in a -20°C refrigerator.

[0105] 10. HIV, HCV, HDV single-cut enzyme: Use SspI-HF single-cut enzyme to linearize HIV / HCV / HDV plasmids to obtain linearized plasmids, as follows:

[0106] SspI-HF single-cutting enzyme system

[0107] Reaction components Dosage (μl) DNA 5 10XNEBuffer 5 RestrictionEnzyme 1 Nuclease-freeWater 39 Total 50

[0108] Reaction conditions: Prepare the system on ice, centrifuge briefly, incubate at 37°C for 15 min, and heat inactivate at 65°C for 2 min.

[0109] Verify the linearized plasmid: After the enzyme digestion, take 5 μl for gel electrophoresis and use the plasmid DNA as a control.

[0110] 11. In vitro transcription: using RiboMAX TM Large Scale RNA Production Systems—T7 was used for in vitro transcription, and HIV / HCV / HDV linearized plasmids were used as templates to obtain HIV-RNA, HCV-RNA, and HDV-RNA. The details are as follows:

[0111] In vitro transcription reaction system

[0112]

[0113]

[0114] Reaction conditions: Mix gently by pipetting, incubate at 37℃ for 4h

[0115] After the transcription reaction was completed, proceed directly to the DNase step by adding 1 μ / μl RQ1 RNase-Free DNase to the reaction system and incubate at 37°C for 15 min.

[0116] 12. Purification of in vitro transcription products: Purify the in vitro transcription products using the phenol-chloroform extraction method to obtain purified in vitro transcribed RNA. The steps are as follows:

[0117] (1) Preparation: Prepare the reagents phenol / chloroform / isoamyl alcohol (25:24:1), phenol / isoamyl alcohol (24:1), 70% ethanol, anhydrous ethanol, 3M sodium acetate, and isopropanol. Centrifuge at 4°C. Then, add ddH2O to make up to 100 μl of the in vitro transcription product.

[0118] (2) Add 100 μl of phenol / chloroform / isoamyl alcohol (25:24:1) to the 1.5 ml EP tube containing the in vitro transcription product, shake to mix, and centrifuge at 12,000 rpm at 4°C for 2 min.

[0119] (3) Carefully transfer the supernatant from the EP tube to a new 1.5 ml EP tube, add an equal volume of chloroform / isoamyl alcohol (24:1), shake and mix, and centrifuge at 12000 rpm at 4°C for 2 min; Carefully transfer the supernatant from the EP tube to a new 1.5 ml EP tube, add 1 / 10 volume of 3 M sodium acetate and an equal volume of isopropanol, shake and mix, place in a -20°C refrigerator for 5 min, and centrifuge at 15000 rpm at 4°C for 10 min.

[0120] (4) Remove the supernatant, add 1 ml of 70% ethanol, mix by inversion 10 times, and centrifuge at 4°C 15000 rpm for 10 min; remove the ethanol, add 1 ml of anhydrous ethanol, mix by inversion 10 times, and centrifuge at 4°C 15000 rpm for 10 min; discard the supernatant, invert the EP tube on paper in the safety cabinet for 5 minutes until the ethanol evaporates; add 20 μl of eluent to dissolve the precipitate, and store in a -80°C refrigerator.

[0121] 13. Determination and dilution of standard RNA and DNA concentration: Use UV spectrophotometer to determine RNA / DNA concentration;

[0122] (1) Calculation of copy number

[0123] RNA: Calculation formula based on concentration and copy number conversion:

[0124] copies / ul=concentration(ng / ul)×10 -9 ×6.02×10 23 (copies / mol) / (RNA length × 330)

[0125] In this way, the standard positive RNA copy number can be calculated;

[0126] DNA: Calculation formula based on concentration and copy number conversion:

[0127] copies / ul=concentration(ng / ul)×10 -9 ×6.02×10 23 (copies / mol) / (DNA length × 660)

[0128] In this way, the standard positive plasmid copy number can be calculated.

[0129] (2) Standard dilution: dilute the plasmid standard and RNA standard to 4×10 9 10 copies / μl, mix equal volumes, take 3μl of the mixed standard, add 27μl ddH2O, dilute 10 times, flick the bottom of the tube with your finger to mix, centrifuge twice, repeat this step until the dilution is 10 1 copies / μl, stored at -80℃ for future use.

[0130] S4. Optimization of TaqMan multiplex RT-qPCR reaction conditions (only required when establishing the method for the first time): Use specific primers and probe combinations to perform PCR detection on plasmid standards and RNA standards of HIV, HBV, HCV, and HDV, and optimize the reaction system and reaction conditions;

[0131] Furthermore, the reaction system and reaction conditions optimized in S4 are as follows: primers, probes, annealing temperature and reverse transcription temperature of four viruses in a 25 μL reaction system;

[0132] The upstream and downstream primers (10 μmol / L) were used in amounts of 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, and 0.7 μL;

[0133] The probe (10 μmol / L) was used in amounts of 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, and 0.7 μL;

[0134] The annealing temperatures are optimized at 56°C, 58°C, 60°C, and 62°C;

[0135] The reverse transcription temperature is optimized at 48°C, 50°C, 52°C, and 54°C;

[0136] The number of cycles of the multiplex TaqMan fluorescent quantitative PCR was 40 times.

[0137] S5. Construct a standard curve: Mix equal volumes of the four standards of HIV, HBV, HCV, and HDV, and select 10 9 -10 4 The mixed standard of 1000 copies / μl was used as the template, and ddH2O was used as the negative template. TaqMan multiple RT-qPCR amplification was performed according to the optimized reaction system and reaction conditions. The amplification efficiency was analyzed by real-time fluorescence quantitative instrument matching software. The standard curve was drawn according to the linear relationship between the logarithm of the starting copy number of the standard and the CT value of the four viruses (such as the standard curve Figure 1 , amplification curve Figure 2 );

[0138] And the linear regression equation is shown in the following table, and the correlation coefficient of the fitted linear equation is R 2 >0.990, indicating that the standard curve has good linearity.

[0139]

[0140] S6. Test of sensitivity, specificity, and repeatability of multiplex TaqMan fluorescence quantitative PCR (only required when establishing the method for the first time);

[0141] Furthermore, the sensitivity test of TaqMan multiplex RT-qPCR in S6 specifically includes: mixing equal volumes of four standards of HIV, HBV, HCV, and HDV, and selecting 10 8 ~10 2 The mixed standard of 1000 copies / μl was used as template and ddH2O as negative control. TaqMan multiplex RT-qPCR amplification was performed according to the optimized reaction system and reaction conditions to evaluate the sensitivity of the method (such as Figure 3 ).

[0142] Conclusion: If Figure 3 It can be seen that the lowest copy number detected is 1×10 3 copies / μL, HBV is 1×10 2 copies / μL, HCV is 1×10 3 copies / μL, HDV is 1×10 3 copies / μL indicates that the method has good sensitivity at this plasmid concentration.

[0143] Furthermore, the specificity test of the TaqMan multiplex RT-qPCR in the S6 specifically includes: in the HIV, HBV, HCV, and HDV TaqMan multiplex RT-qPCR detection method, using HIV, HCV, HDV RNA and HBV plasmid as templates, using common flavivirus viruses dengue virus (DENV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), and iris virus (HT5K), ddH2O as a negative control, and performing TaqMan multiplex RT-qPCR amplification according to the optimized reaction system and reaction conditions to evaluate the specificity of the method.

[0144] Conclusion: If Figure 4 It can be seen that only the templates of HIV / HBV / HCV / HDV show fluorescence signals, indicating that this method has good specificity.

[0145] Furthermore, the repeatability test of TaqMan multiplex RT-qPCR in S6 specifically includes: in the HIV, HBV, HCV, HDV TaqMan multiplex RT-qPCR detection method, using HIV, HCV, HDV RNA and HBV plasmid as templates, ddH2O as a negative control, according to the optimized reaction system and reaction conditions, select 10 9 copy / μl, 10 7 copy / μl, 10 5The experiment was repeated within and between groups with three gradients of 1000 copies / μl. The average value and coefficient of variation (CV) of the obtained Cq values ​​were calculated in Excel to evaluate the repeatability of the method.

[0146] Table 11 Intra-group coefficient of variation of TaqMan multiplex RT-qPCR repeatability test

[0147]

[0148] Table 12 Coefficient of variation between groups in TaqMan multiplex RT-qPCR repeatability test

[0149]

[0150] Conclusion: As shown in the table above, according to the analysis of intra-group and inter-group repeatability results, the coefficient of variation (CV) of each group was less than 4.97%, indicating that the TaqMan multiplex RT-qPCR method established in this experiment had good stability.

[0151] S7, sample testing: After the accuracy of the TaqMan multiplex RT-qPCR detection method for four blood-borne diseases has been verified by S6, PCR testing can be performed on the four virus samples, and the test results can be determined based on the test data;

[0152] Comparison of real-time fluorescence quantitative PCR and conventional PCR in detecting clinical samples

[0153]

[0154] Conclusion: Among the 43 samples tested, the TaqMan multiplex RT-qPCR method detected 5 HIV cases, 11 HBV cases, 24 HCV cases, 0 HDV cases, 3 HIV+HCV cases, 1 HIV+HBV case, and 2 HBV+HCV cases. The sample detection rates were all higher than those of the common PCR, indicating that the TaqMan multiplex RT-qPCR method established in this experiment had a good detection rate.

[0155] The test results are determined by:

[0156] Positive conditions are: CT value of test result <35;

[0157] Negative condition: CT value ≥35.

[0158] Based on the above, the present invention also provides four blood-borne disease TaqMan multiplex RT-qPCR detection kits, the kits include: Takara One Step PrimeScript TMIII RT-qPCR Mix (2x), ddH2O, four blood-borne virus mixed primers, four blood-borne virus mixed probes, positive control: four virus standard mixed samples, negative control: ddH2O;

[0159] Among them, the sequences of the four blood-borne virus mixed primers are shown as SEQ ID No.5 to SEQ ID No.12, and the sequences of the four blood-borne virus mixed probes are shown as SEQ ID No.13 to SEQ ID No.16 respectively.

[0160]

[0161]

[0162] Furthermore, the positive control is a mixed standard containing four blood-borne viruses HIV, HBV, HCV, and HDV, and the concentration of the positive control is 10 9 ~10 4 copy / μl.

[0163] Based on the above, the present invention also provides the use of four blood-borne disease TaqMan multiplex RT-qPCR detection kits in detecting one or more of HIV, HBV, HCV, and HDV.

[0164] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0165] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. TaqMan multiplex RT-qPCR detection method for four blood-borne diseases, characterized in that: The specific steps include: S1. Extraction of viral RNA from samples: RNA of four strains of HIV, HBV, HCV, and HDV was extracted using TIANamp Virus RNAKit to obtain viral RNA samples; S2. Specific primer and probe design: Design specific primers and probes according to HIV, HBV, HCV, and HDV strains; Wherein, the sequences of the primers are shown in SEQ ID No.5 to SEQ ID No.12, and the sequences of the probes are shown in SEQ ID No.13 to SEQ ID No.16; S3. Preparation of standard templates: Prepare 19T-HBV plasmid standards and HIV, HCV, and HDV in vitro transcribed RNA standards based on viral RNA samples, and use them as standards; S4. Optimization of TaqMan multiplex RT-qPCR reaction conditions: Use specific primers and probe combinations to perform TaqMan multiplex RT-qPCR detection on plasmid standards and RNA standards of HIV, HBV, HCV, and HDV, and optimize the reaction system and reaction conditions; S5. Construct a standard curve: Mix equal volumes of the four standards of HIV, HBV, HCV, and HDV, and select 10 9 -10 4 The mixed standard of 1000 copies / μl was used as the template, and ddH2O was used as the negative template. TaqMan multiplex RT-qPCR amplification was performed according to the optimized reaction system and reaction conditions. The amplification efficiency was analyzed by real-time fluorescence quantitative instrument matching software, and the standard curve was drawn according to the linear relationship between the logarithm of the starting copy number of the standard and the CT value of the four viruses. S6, sensitivity, specificity, and repeatability test of TaqMan multiplex RT-qPCR; S7, sample testing: After the accuracy of the TaqMan multiplex RT-qPCR detection method for four blood-borne diseases has been verified by S6, TaqMan multiplex RT-qPCR detection can be performed on the four virus samples, and the test results can be judged based on the test data; The test results are determined by: Positive conditions are: CT value of test result <35; Negative condition: CT value ≥35.

2. The method for detecting four blood-borne diseases according to claim 1, characterized in that: The reaction system and reaction conditions optimized in the S4 are as follows: primers, probes, annealing temperature and reverse transcription temperature of four viruses in a 25 μL reaction system; The upstream and downstream primers (10 μmol / L) were used in amounts of 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, and 0.7 μL; The probe (10 μmol / L) was used in amounts of 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, and 0.7 μL; The annealing temperatures are optimized at 56°C, 58°C, 60°C, and 62°C; The reverse transcription temperature is optimized at 48°C, 50°C, 52°C, and 54°C; The number of cycles of the TaqMan multiplex RT-qPCR was 40 times.

3. The TaqMan multiplex RT-qPCR detection method for four blood-borne diseases according to claim 1, characterized in that: The sensitivity test of TaqMan multiplex RT-qPCR in S6 specifically includes: mixing equal volumes of four standards of HIV, HBV, HCV, and HDV, selecting 10 8 ~10 2 The mixed standard of 1000 copy / μL was used as template and ddH2O as negative control. TaqMan multiplex RT-qPCR amplification was performed according to the optimized reaction system and reaction conditions to evaluate the sensitivity of the method.

4. The TaqMan multiplex RT-qPCR detection method for four blood-borne diseases according to claim 1, characterized in that: The specificity test of TaqMan multiplex RT-qPCR in S6 specifically includes: in the HIV, HBV, HCV, HDV TaqMan quadruple RT-qPCR detection method, using HIV, HCV, HDV RNA and HBV plasmid as templates, using common flavivirus viruses dengue virus (DENV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), iridovirus (HT5K), ddH2O as a negative control, and performing TaqMan multiplex RT-qPCR amplification according to the optimized reaction system and reaction conditions to evaluate the specificity of the method.

5. The TaqMan multiplex RT-qPCR detection method for four blood-borne diseases according to claim 1, characterized in that: The repeatability test of TaqMan multiplex RT-qPCR in S6 specifically includes: in the HIV, HBV, HCV, HDV TaqMan multiplex RT-qPCR detection method, using HIV, HCV, HDV RNA and HBV plasmid as templates, ddH2O as a negative control, according to the optimized reaction system and reaction conditions, select 10 9 copy / μl, 10 7 copy / μl, 10 5 The experiment was repeated within and between groups with three gradients of 1:1 copy / μl. The mean value and coefficient of variation (CV) of the obtained Cq values ​​were calculated in Excel to evaluate the repeatability of the method.

6. Four blood-borne disease TaqMan multiplex RT-qPCR detection kits, characterized in that: Kit includes: Takara One Step PrimeScript TM III RT-qPCRMix (2x), ddH2O, four blood-borne virus mixed primers, four blood-borne virus mixed probes, positive control: four virus standard mixed samples and negative control: ddH2O; Among them, the sequences of the four blood-borne virus mixed primers are shown as SEQ ID No.5 to SEQ ID No.12, and the sequences of the four blood-borne virus mixed probes are shown as SEQ ID No.13 to SEQ ID No.16 respectively.

7. The TaqMan multiplex RT-qPCR detection kit for four blood-borne diseases according to claim 6, characterized in that: The positive control is a mixed standard containing four blood-borne viruses HIV, HBV, HCV, and HDV, and the concentration of the positive control is 10 9 ~10 4 copy / μl.

8. Use of the TaqMan multiplex RT-qPCR detection kit for four blood-borne diseases according to any one of claims 6 to 7 in detecting one or more of HIV, HBV, HCV, and HDV.

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