WO2018035860A1 - 同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的试剂盒及方法 - Google Patents

同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的试剂盒及方法 Download PDF

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WO2018035860A1
WO2018035860A1 PCT/CN2016/096964 CN2016096964W WO2018035860A1 WO 2018035860 A1 WO2018035860 A1 WO 2018035860A1 CN 2016096964 W CN2016096964 W CN 2016096964W WO 2018035860 A1 WO2018035860 A1 WO 2018035860A1
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seq
blood
qpcr
virus
kit
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PCT/CN2016/096964
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English (en)
French (fr)
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李展平
黄娟娟
涂义芳
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旭基科技股份有限公司
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Priority to PCT/CN2016/096964 priority Critical patent/WO2018035860A1/zh
Priority to CN201680084986.2A priority patent/CN109196124A/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

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  • the invention provides a kit for simultaneously detecting and quantitatively analyzing multiple Taqman probe qPCRs of four blood-borne viruses; in particular, providing a method for simultaneously detecting and quantifying hepatitis B virus (HBV) ), hepatitis C virus (HCV), human immunodeficiency virus type 1 (HIV1), and human T-cell lymphoma/leukemia virus type 1 (human T-cell lymphoma/leukemia virus type 1) , HTLV1)
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HCV1 human immunodeficiency virus type 1
  • HCV1 human T-cell lymphoma/leukemia virus type 1
  • HTLV1 human T-cell lymphoma/leukemia virus type 1
  • Blood-borne viruses are transmitted mainly by exposure to blood or blood products, exposure to certain body tissues and body fluids, sexual contact and diseased mothers.
  • Common blood-borne viruses include hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus type 1 (HIV1), human T-cell lymphoma/leukemia type 1 (HTLV1), and other viruses.
  • HBV and HCV infections are the most common and severe form of liver disease, affecting more than 5 million people worldwide.
  • Taiwan 90% of adults have been infected with HBV; about 15% to 20% (about 3 million) are carriers of hepatitis B virus surface antigen (HBsAg).
  • Taiwan Since 1985, Taiwan has implemented a national neonatal vaccination against hepatitis B vaccine to significantly reduce the number of viral carriers of hepatitis B virus surface antigen (HBsAg).
  • Long-term illnesses that infect HBV include cirrhosis and liver cancer; death from hepatitis and cirrhosis is the sixth in all deaths, and in Taiwan, liver cancer is second only to lung cancer, the second most common cancer. cause of death.
  • about 300,000 people in Taiwan are carriers of HCV, about 5% of the total population or 1/10 of HBV carriers.
  • the human lentivirinae group is a combination of HIV-1 and HIV-2, which is associated with severe deficiencies and acquired immunodeficiency syndrome (AIDS). Individuals infected with these viruses are highly likely to develop the disease, and the average time to onset of HIV1 is about 10.7 years. Delayed treatment or no HIV test results or Delaying entry into HIV-positive care is a common health problem.
  • AIDS acquired immunodeficiency syndrome
  • HTLV-1 and HTLV-2 Human oncogenic viruses
  • human T-lymphotropic viruses which are associated with benign and tumor lymphoproliferative disorders, various autoimmune diseases, and immunity. Defect related. However, most individuals who are infected with both viruses can remain asymptomatic.
  • the United States Food and Drug Administration recommends screening for HTLV1 due to the possibility of infection with the virus. In the United States, the seroprevalence of HTLV1 infection by blood donors is within 0.025%. Quantification of HTLV1 RNA by qRT-PCR in serum or plasma is the most direct method for detecting viral infection, replication and therapeutic effects.
  • Taiwan blood bank used ELISA to screen 30,000 blood samples by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the results showed that the positive rate of HBV was 1.09%, the positive rate of HCV was 0.28%, and HIV1/1/ The positive rate of 2 was 0.06%, the positive rate of HTLV was 0.05%, and the positive rate of Treponema pallidum (TP) was further detected to be 0.07%.
  • NAT nucleic acid amplification technology
  • the positive rate of nucleic acid amplification technology will be higher than that of the above ELISA detection method due to the empty window period of various viruses.
  • blood is in great demand. About 38,000 people need red blood cells during the day, including accident victims, patients undergoing surgery, and receiving leukemia, cancer, or other diseases such as sickle cell disease. There are more than 26 million transfusion treatments per year for patients treated with thalassemia.
  • Taiwan blood bank has been screened for HBV, HCV, HIV 1/2, and HTLV1/2 by enzyme-linked immunosorbent assay (ELISA).
  • the detection of HBV is a hepatitis B virus surface antigen, while other viruses (HCV, HIV, and HTLV) are tested for corresponding antigens and antibodies.
  • the detection of ELISA has the disadvantages of low sensitivity, cross-reactivity and the possibility of false negatives.
  • the empty window period of the HCV antibody is 70 days
  • the empty window period of the HIV antibody is 22 days
  • the empty window period of the hepatitis B virus surface antigen is 56 days. Since current serum screening tests fail to detect the infectious window of an infected donor or fail to detect the various antigenic types of these viruses, there is a risk of pathogenic viral transmission.
  • NAT Nucleic Acid Amplification
  • kits containing a primer set and a probe set which can be applied to a clinical blood sample and can simultaneously detect and quantify four blood-borne viruses having various genotypes in a single test tube.
  • the present invention provides a kit for simultaneous detection and quantification of multiple Taqman probe qPCRs for four blood-borne viruses, comprising: a group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, SEQ ID NO a group consisting of: 4 to SEQ ID NO: 6, a group consisting of SEQ ID NO: 7 to SEQ ID NO: 9, and a group consisting of SEQ ID NO: 10 to SEQ ID NO: 12; wherein SEQ ID NO : 1 to SEQ ID NO: 3 for detection of hepatitis B virus; SEQ ID NO: 4 to SEQ ID NO: 6 for detection of hepatitis C virus; and SEQ ID NO: 7 to SEQ ID NO: 9 for detection of human Immunodeficiency virus type 1; and SEQ ID NO: 10 to SEQ ID NO: 12 were used to detect human T cell lymphoma/leukemia type 1 (HTLV1), and the qPCR was performed in a single reaction mixture.
  • HTLV1 human T cell lymphoma/
  • the present invention further provides a kit for detecting and quantifying a Taqman probe qPCR of hepatitis C virus, comprising: a group consisting of SEQ ID NO: 4 to SEQ ID NO: 6.
  • the present invention further provides a kit for detecting and quantifying Taqman probe qPCR of human immunodeficiency virus type 1 comprising: a group consisting of SEQ ID NO: 7 to SEQ ID NO: 9.
  • the invention also provides a method for simultaneous detection and quantification of multiple Taqman probe qPCRs for four blood-borne viruses, comprising: (a) providing a biological sample; (b) providing a primer set comprising SEQ ID NO: a group consisting of SEQ ID NO: 2; a group consisting of SEQ ID NO: 4 and SEQ ID NO: 5; a group consisting of SEQ ID NO: 7 and SEQ ID NO: 8; and SEQ ID NO: 10.
  • SEQ ID NO: 11 a group consisting of SEQ ID NO: 11; (c) providing a probe set comprising SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12; (d) using the a primer set, the probe set, and the biological sample are subjected to qPCR; and (e) observing the generated reaction product to determine whether the blood-borne virus is present in the biological sample and quantifying the blood-borne virus, wherein SEQ ID NO: 1 to SEQ ID NO: 3 for detection of hepatitis B virus (HBV); SEQ ID NO: 4 to SEQ ID NO: 6 for detection of hepatitis C virus (HCV); SEQ ID NO: 7 to SEQ ID NO:9 for detection of human immunodeficiency virus type 1 (HIV1); and SEQ ID NO:10 to SEQ ID NO:12 for detection of human T cell lymph A tumor/leukemia type 1 (HTLV1), and the qPCR was carried out in a single reaction mixture.
  • HBV
  • SEQ ID NO: 3 SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12 have a fluorescent dye at the 5' end and a fluorescent inhibitory molecule at the 3' end needle.
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10 And SEQ ID NO: 11 are primers.
  • the present invention provides a kit for simultaneously detecting and quantifying various genotypes of four blood-borne viruses in a clinical single blood sample tube, wherein the blood-borne virus includes hepatitis B virus (HBV), Hepatitis C virus (HCV), human immunodeficiency virus type 1 (HIV1), and human T cell lymphoma/leukemia type 1 (HTLV1).
  • the kit containing the primer and the probe of the invention can be used for detecting the high-throughput detection mode of the 96-well plate within 1.5 hours, and the sealed test tube form can reduce the possibility of cross-contamination and has high sensitivity and Specificity. Therefore, the reagent of the present invention
  • the kit can be used for blood screening of blood donors, detection of blood products, detection of cord blood stem cells, evaluation of the efficacy of anti-viral drugs, development of anti-viral drugs, and other uses.
  • 1A to 1E are quantitative amplification amplification results and standard curves of a kit for simultaneously detecting and quantifying multiple Taqman probe qPCR (M-tp-qPCR) of four blood-borne viruses.
  • the quantitative standard (QS) copy number is 10 3 to 10 7 ;
  • the upper graph of Figures 1A to 1D, and Figure 1E are the amplification patterns of the four viruses and internal control components (IC) of HBV, HCV, HIV1, and HTLV1.
  • the lower panel of Figures 1A-1D is a standard curve for four viruses, HBV, HCV, HIV1, and HTLV1.
  • M-tp-qPCR a kit for simultaneously detecting and quantifying multiple Taqman probes qPCR (M-tp-qPCR) of four blood-borne viruses according to the present invention, and black pointers indicate PCR-specific products of four viruses;
  • the white pointer represents the PCR product of the internal control component (IC).
  • FIG. 4A to 4D are reproducibility of a kit for simultaneously detecting and quantifying multiple Taqman probe qPCR (M-tp-qPCR) of four blood-borne viruses, (Fig. 4A) HCV, (Fig. 4B) HBV, (Fig. 4C) HIV1, and (Fig. 4D) HTLV1.
  • M-tp-qPCR multiple Taqman probe qPCR
  • 5A to 5D are the sensitivity of the kit for simultaneously detecting and quantifying multiple Taqman probe qPCR (M-tp-qPCR) of four blood-borne viruses, (Fig. 5A) HCV, (Fig. 5B) HBV, (Fig. 5C) HIV1, and (Fig. 5D) HTLV1.
  • M-tp-qPCR multiple Taqman probe qPCR
  • FIG. 6A to 6D are multiple simultaneous detection and quantitative analysis of four blood-borne viruses Dynamic range analysis of kits for Taqman probe qPCR (M-tp-qPCR), (Fig. 6A) HCV, (Fig. 6B) HBV, (Fig. 6C) HIV1, and (Fig. 6D) HTLV1.
  • Figure 14 shows the effect of nucleotide variation of the HTLV1 primer of the present invention on qPCR specificity
  • 3'-G represents a kit for Taqman probe qPCR of the present invention
  • 3'-A represents A single nucleotide at the 3' end of the forward primer sequence (SEQ ID NO: 10) of the HTLV1 virus was altered.
  • the kit for simultaneous detection and quantitative analysis of four blood-borne viruses of multiple Taqman probes qPCR can be used for simultaneously or separately detecting and quantifying four blood-borne viruses in vitro, including type B. Hepatitis virus, hepatitis C virus, human immunodeficiency virus type 1, and human T cell lymphoma/leukemia type 1.
  • the kit of the present invention can be used for blood screening of blood donors, detection of blood products, detection of cord blood stem cells, evaluation of the efficacy of anti-viral drugs, development of anti-viral drugs, and other uses.
  • the kit of the invention has extremely high polymerase chain reaction (PCR) sensitivity, quantitative analysis accuracy, specificity of Taqman probe hybridization, hydrolyzability of Taq DNA polymerase 5' nuclease activity and wide range The scope of detection.
  • the kit for simultaneously detecting and quantifying multiple blood-borne viruses of multiple Taqman probes qPCR is suitable for two-step quantitative reverse transcription PCR detection method: first step, random The hexamer reverse transcribes RNA viruses (including HCV, HIV, and HTLV) into cDNA; and in the second step, the purified HBV DNA virus samples are subjected to multiplex Taqman probe qPCR.
  • the Taqman probe uses FAM/BHQ1 (HCVtp-313R-FAM), HEX/BHQ1 (HBVtp-395R-HEX), CAL Fluorescent Red 610/BHQ2 (HIV1papPEtp-610), and Quasar 670/BHQ2 (HTLV1-73-tp-, respectively). 670) and Quasar 705/BHQ2 (Qbeta tp-705) markers, each paired with a fluorescent dye and a fluorescent inhibitory molecule.
  • the BioRad iQ5 real-time thermal cycler has five sets of excitation filter banks and radiation filter banks, replacing the original TAMRA filter set (channel 4) with a 705 nm filter bank. The new combination has a minimum overlap of excitation/emission wavelengths and 5 fluorescent stains can be used simultaneously.
  • HBV A No. X70185
  • HBV B No. D00331)
  • HBV C No. X01587)
  • HBV D No. X72702
  • HBV E No. X75664
  • HBV F No. X75663
  • HBV G No. AF405706
  • HBV H No. EF157291
  • HCV 1a No. M67463
  • HCV 1b No. AB016785
  • HCV 2a No. AB047639
  • HCV 2b No.
  • HCV 3a No. AF046866
  • HCV 3b No. D49374
  • HCV 4a No. D45193, Y11604
  • HCV 5a No. D50466, Y13184
  • HCV 6a No. D88469, AY859526
  • HIV1 number NC_001802
  • HTLV1 number NC_001436
  • the present invention purifies serum or plasma samples using a viral DNA/RNA purification kit (Ambergene, Taiwan).
  • Serum can be collected using a serum separation tube; leukocytes are collected using sterile EDTA or citrate blood collection tubes, and samples containing anticoagulant drugs are not suitable for sample purification.
  • the blood collection tube must be centrifuged at 3,000 rpm for 10 minutes at room temperature to collect serum or plasma; then the serum or plasma is pipetted to the polypropylene test.
  • Intratube used as viral DNA/RNA purification.
  • serum or plasma can be stored in a -20 ° C freezer for later use.
  • Leukocytes must be delivered at 2 to 20 °C within six hours of collection; serum or plasma can be shipped at 2 to 8 °C or shipped frozen.
  • the present invention uses a MagBead virus DNA/RNA kit (Ambergene, Taiwan) or a silicated membrane column for viral purification DNA/RNA Kit (Ambergene, Taiwan).
  • the viral DNA/RNA was purified from 0.2 mL of serum or plasma samples according to the manufacturer's manual, and finally the viral DNA/RNA was dissolved in 50 ⁇ L of RNase-free and DNase-free solutions.
  • the viral DNA/RNA was further precipitated with isopropanol and concentrated to 25 ⁇ L with a 0.3 M NaOAc pH 5.2 solution. 5 ⁇ L of viral DNA/RNA was subjected to cDNA synthesis using a random hexamer, and 1 ⁇ L of cDNA was used for each PCR analysis.
  • the total reaction volume of cDNA synthesis was 10 ⁇ L, containing 1 ⁇ L of purified viral DNA/RNA, 1 ⁇ g of random hexamer primer, 0.5 ⁇ L of 10 mM dNTPs, 2 ⁇ L of 5 ⁇ 1 strand buffer (5 ⁇ cDNA1 st strand synthesis buffer) (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl 2 ), 0.5 ⁇ L 0.1 M DTT, 0.1 to 0.25 ⁇ L RNase inhibitor (40 units/ ⁇ L), and 0.1 to 0.25 ⁇ L MMLV RT (200 U/ ⁇ L, Ambergene, Taiwan) .
  • the reaction solution was first reacted at 25 ° C for 5 minutes to bind the hexamer primer to the RNA template, and the first strand of cDNA was reacted at 30 ° C for 10 minutes, at 40 ° C for 40 minutes, and at 95 ° C for 5 minutes. synthesis. In an ideal state, more than one RNA target sequence is simultaneously reverse transcribed in the sample.
  • the completed cDNA product was placed on ice for multiplex Taqman probe qPCR (M-tp-qPCR) or single Taqman probe qPCR; or the cDNA product was stored in a -20 ° C refrigerator for later use.
  • the invention utilizes five filter membrane groups (FAM-495 (excitation) / 520 (emission), HEX-535 (excitation) / 556 (emission), CAL fluorescence red 610-590 (excitation) / 610 (emission), Quasar 670-647 (excitation) / 667 (emission), Quasar 705-690 (excitation) / 705 (emission) BioRad iQ5 real-time thermal cycler.
  • the method of the multiplex Taqman probe qPCR of the present invention has a total reaction volume of 20 ⁇ L, including 20 mM Tris-HCl pH 8.8, 10 mM KCl, 3 mM MgCl 2 , 0.1% Triton X-100, 0.1 mg/mL BSA, 0.25 mM per dNTP, 0.4 mM per primer, 0.25 mM per Taqman probe and 2 units of heat Start Taq DNA polymerase (heated to above 42 ° C for 1 minute for activation) (Ambergene, Taiwan).
  • kits for simultaneously detecting and quantifying multiple Taqman probe qPCR M-tp-qPCR
  • M-tp-qPCR multiple Taqman probe qPCR
  • the Y intercept can be used as one of the parameters for monitoring the sensitivity of the analysis.
  • a Y value of more than 40 indicates a slow response and a low analytical sensitivity, and a Y value of less than 40 indicates a faster response and usually is not
  • the correct template replicates the background value of the calculated or non-specific PCR product.
  • Higher magnesium ion concentrations increase the binding of primer hybridization, but also increase the production of non-specific products and the formation of primer-primer dimers.
  • the kit for simultaneous detection and quantification of multiple blood-borne viruses of multiple Taqman probes qPCR (M-tp-qPCR) to HBV, HCV
  • M-tp-qPCR multiple Taqman probes qPCR
  • the specificity of the four viruses, HIV1, and HTLV1 were excellent.
  • the PCR efficiencies of HBV, HCV, HIV1, and HTLV1 were 96.9, 95, 96.9, and 113.2, respectively.
  • the linear relationship of the analysis was from 1.000 to 0.997. They are - 3.399, -3.448, -3.399, and -3.014, respectively, and the sensitivity is as high as 10 1 to 10 2 copy numbers.
  • kits for simultaneous detection and quantification of multiple blood-borne viruses of multiple Taqman probes qPCR of the present invention can be used for detecting cDNA, containing different initial concentrations, and containing four blood sources.
  • the DNA template of the viral sequence is suitable for the analysis method of the present invention; the kit of multiple Taqman probe qPCR (M-tp-qPCR) or single Taqman probe qPCR is respectively used to quantify the standard 10 3 to 10 7 or 10 2 to 10 7 copy number is performed.
  • a total of 477 clinical blood samples were used in the present invention, and viral DNA was purified using MagBead virus DNA/RNA kit (Ambergene, Taiwan) or Column Viral DNA/RNA Kit (Ambergene, Taiwan). /RNA.
  • MagBead virus DNA/RNA kit Ambergene, Taiwan
  • Column Viral DNA/RNA Kit Ambergene, Taiwan
  • the samples were tested and identified by quantitative PCR, real-time SYBR green PCR, Northern blotting, and DNA sequencing.
  • kits of simultaneous detection and quantitative analysis of four blood-borne viruses for multiple Taqman probe qPCR (M-tp-qPCR)
  • the identification of HBV, HCV, HIV1, and HTLV1 viruses is 100% and the above Previous detection techniques are consistent.
  • these sample mixtures contained various genotype panel standards (HBV A to H genotype, HCV 1 to 6 genotype, HIV1, and HTLV1).
  • the kit for simultaneously detecting and quantifying the multiple Taqman probe qPCR (M-tp-qPCR) of four blood-borne viruses is 100%, which is consistent with previous detection techniques, and can be used for detecting the above four viruses. All genotypes.
  • kits quantification of the multiplex multiplexed Taqman probe qPCR for simultaneous detection and quantification of four blood-borne viruses of the present invention further provide the copy number of each positive reaction virus sample.
  • the copy number was distributed in the range of 10 2 to 10 7 copies/mL, and the average copy number per ml of blood samples was 10 4 to 10 5 .
  • Copy number/mL 91 HBV PCR positive samples, copy number distribution in the range of 10 2 to 10 8 copies / mL, the average copy number per ml of blood samples is 10 5 copies / mL; 20 HIV1 For PCR-positive samples, the copy number is distributed in the range of ⁇ 10 2 to 10 4 copies/mL, and the average copy number per ml of blood sample is 10 3 copies/mL; 19 samples of HTLV1 PCR positive reaction, copy The number distribution is in the range of ⁇ 10 2 to 10 4 copies/mL, and the average copy number per ml of blood sample is 10 3 copies/mL.
  • the present invention utilizes six replicate samples of a control component plasmid quantification standard (QS) containing a DNA insert sequence for amplification to confirm reproducibility. Enter each copy number (from 10 0 to 10 6 ) to obtain the Ct value, the average value of the Ct value, and thus the standard deviation (SD).
  • QS control component plasmid quantification standard
  • the results shown in FIG. 4A to 4D, the well shown in Table 5, the present invention is by HBV, HCV, HIV1, HTLV1 template and input 100 to 106 are respectively the copy number of the standard deviation of Ct values obtained (from 100 to 106) (SD) is 0.21 to 2.50, 0.19 to 2.1, 0.27 to 2.62, and 0.26 to 2.52.
  • the present invention utilizes serial dilutions of ten replicate samples containing the DNA Control Group Plasmid Quantitative Standard (QS) for amplification to confirm sensitivity (from 10 0 to 10 6 ) and calculate a positive detection rate.
  • QS DNA Control Group Plasmid Quantitative Standard
  • Figures 5A to 5D, and Table 6 all HBV, HCV, HIV1, and HTLV1 viruses have detection sensitivities as high as 10 to 10 2 copies.
  • the present invention serially dilutes four viral control component plasmid DNAs from 10 1 to 10 9 to confirm the dynamic range and quantitative analysis of the four viruses. As shown in Figures 6A-6D and Table 5, all four viruses have a wide dynamic range from 10 1 to 109 . All four virus analyses showed excellent potency (slope from -3.005 to -3.4133), excellent linearity (R 2 from 0.999 to 0.9996), and excellent amplification (Y intercept from 38.031 to 39.433) .
  • the present invention analyzes three replicate samples, and uses the kit for simultaneous detection and quantification of multiple blood-borne viruses of multiple Taqman probes qPCR (M-tp-qPCR) to obtain copy number and standard Serial dilutions of copy number.
  • M-tp-qPCR multiplex multiplexed Taqman probe qPCR
  • FIG. 7A to FIG. 7D the copy number and standard dilution of the multiplex multiplexed Taqman probe qPCR (M-tp-qPCR) of the four blood-borne viruses simultaneously and quantitatively analyzed by the present invention are serially diluted with the standard.
  • the copy number has a highly linear relationship (R 2 ), where the HCV linear relationship (R 2 ) is 0.9732 (from 10 1 to 10 5 ), the HBV linear relationship (R 2 ) is 0.9793 (from 10 1 to 10 6 ), HIV1
  • the linear relationship (R 2 ) is 0.9447 (from 10 1 to 10 4 ), the HTLV1 linear relationship (R 2 ) is 0.9788 (from 10 1 to 10 4 ); and the higher copy number is more correlated with lower copy number display. Sexuality and higher reproducibility.
  • Example 3 Comparison of multiplex taqman probe qPCR (M-tp-qPCR) for simultaneous detection and quantification of four blood-borne viruses of the present invention with Taqman probe qPCR of a single blood-borne virus
  • the present invention utilizes a primer set of a single blood-borne virus and a Taqman probe to simultaneously detect and quantify the detection result of the kit of multiple Taqman probe qPCR (M-tp-qPCR) of four blood-borne viruses. Compare.
  • the total volume of the Taqman probe qPCR reaction of a single blood-borne virus is 20 ⁇ L, including 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 3 mM MgCl 2 , 0.1% Triton X-100, 0.1 mg/mL BSA, 0.25 mM per dNTP 0.4 mM per primer, 0.25 mM per Taqman probe, and 2 units of hot-start Taq DNA polymerase (heated to 42 ° C for 1 minute for activation) (Ambergene, Taiwan). Experiments were performed using eight-well strips or 96-well PCR microplates (Bio-Rad, USA).
  • reaction conditions were as follows: reaction at 95 ° C for 1 minute, further reaction at 95 ° C for 1 second, 55 ° C, reaction for 30 seconds, and reaction at 72 ° C for 30 seconds, and the reaction was repeated 40 times.
  • the rest of the methods refer to the detection method of Embodiment 2.
  • the present invention utilizes a primer set of a single blood-borne virus and a Taqman probe to quantify a standard curve of known standard copy number results, as shown in FIGS. 8A to 8D, wherein 57 samples of HCV PCR positive reactions, copy number distribution In the range of 10 to 10 6 copies/mL, the average copy number per ml of blood sample is 1.36 ⁇ 10 5 copies / mL; 91 samples of HBV PCR positive reaction, the copy number is distributed in 10 2 to 10 7 copies In the range of number/mL, the average copy number per ml of blood sample is 4.02 ⁇ 10 6 copies/mL; 20 HIV1 PCR positive samples, the copy number is distributed in the range of ⁇ 10 1 to 10 4 copies/mL Within, the average copy number per ml of blood sample is 1.89 ⁇ 10 4 copies / mL; 19 samples of HTLV1 PCR positive reaction, copy number distribution in the range of ⁇ 10 1 to 10 4 copy number / mL, per ml of blood The average
  • kits of the present invention can detect and quantitatively analyze four blood-borne viruses of HCV, HBV, HIV1, and HTLV1, and can also detect individual viruses by using individual primer sets and probes.
  • Comparative Example 1 Effect of changing the nucleotide specificity of the primer or probe of the present invention on PCR specificity
  • the present invention may be modified by changing the primer of the present invention or The nucleotides of the probes are used to confirm the specificity of the primers or probes of the invention for qPCR reactions.
  • the present invention recognizes the specificity of the primer of the present invention for qPCR by changing the single nucleotide at the 3' end of the forward primer sequence (SEQ ID NO: 1) for detecting the HBV virus to SEQ ID NO: 16.
  • SEQ ID NO: 1 the forward primer sequence for detecting the HBV virus
  • SEQ ID NO: 16 the primer for changing the nucleotide changes the dynamic range to affect the specificity of the PCR, and the higher the number of cycles (Ct) of the primer of the modified nucleotide, the less the initial copy number is confirmed.
  • the kit for detecting and quantifying the Taqman probe qPCR reaction of HBV of the present invention has extremely high sensitivity and specificity.
  • the present invention recognizes that the probe of the present invention is specific for qPCR by changing the single nucleotide of the 12th base C of the intermediate position of the HBV viral probe sequence (SEQ ID NO: 3) to A, G, or T. Sex.
  • the curve of the HBV virus probe sequence (SEQ ID NO: 3) of the present invention it was confirmed by the curve of the HBV virus probe sequence (SEQ ID NO: 3) of the present invention that the HBV virus probe sequence of the present invention is completely matched to the target sequence; the other three single nucleotides A probe that changes (the 12th base changes from C to A, the 12th base changes from C to G, and the 12th base changes from C to T) exhibits a negative reaction result, confirming the use of the present invention.
  • the kit for detecting and quantifying HBV Taqman probe qPCR has a very high spirit Sensitivity and specificity. Broadly speaking, the primers and probes of the present invention have a great influence on sensitivity and characteristics as long as one base is changed, and only the HBV virus is exemplified.
  • the present invention recognizes the specificity of the primer of the present invention for qPCR by changing the single nucleotide C at the 3' end of the forward primer sequence (SEQ ID NO: 4) for detecting the HBV virus to A (SEQ ID NO: 17). .
  • the primer for changing the nucleotide changes the dynamic range to affect the specificity of the PCR, and the higher the number of cycles (Ct) of the primer of the modified nucleotide, the less the initial copy number is confirmed.
  • the kit for detecting and quantifying HCV Taqman probe qPCR of the present invention has extremely high sensitivity and specificity.
  • the present invention recognizes the specificity of the primer of the present invention for qPCR by changing the single nucleotide T at the 3' end of the forward primer sequence (SEQ ID NO: 7) for detecting HIV1 virus to G (SEQ ID NO: 18). .
  • the primer for changing the nucleotide changes the dynamic range and affects the specificity of the PCR.
  • the kit for detecting and quantifying the Taqman probe qPCR reaction of HCV of the present invention has extremely high sensitivity and specificity.
  • the present invention recognizes the specificity of the primer of the present invention for qPCR by changing the single nucleotide G at the 3' end of the forward primer sequence (SEQ ID NO: 10) for detecting the HTLV1 virus to A (SEQ ID NO: 19). .
  • the primer for changing the nucleotide changes the dynamic range to affect the specificity of the PCR, and the higher the number of cycles (Ct) of the primer of the modified nucleotide, the less the initial copy number is confirmed.
  • the kit for detecting and quantifying HCV Taqman probe qPCR of the present invention has extremely high sensitivity and specificity.
  • the kit for simultaneous detection and quantification of four blood-borne viruses of multiple Taqman probes qPCR detects HCV, HBV, HIV1, and HTLV1 viruses.
  • enzyme-linked immunosorbent assays are more effective in reducing detection time, while The ELISA cannot detect a patient with a mutant virus strain and is in an asymptomatic window, and the method of the present invention can be effectively detected.
  • the kit for simultaneous detection and quantification of four blood-borne viruses of multiple Taqman probes qPCR (M-tp-qPCR) for detecting all HBV genotypes (A to H) and all HCV genotypes (1 to 6), HIV1, and HTLV1.
  • the reason why the present invention does not include HIV2 and HTLV2 is that the lower incidence of these two genes is less pathogenic to humans.
  • the method for simultaneously detecting and quantifying the multiple Taqman probe qPCR (M-tp-qPCR) of four blood-borne viruses of the present invention is specifically designed to not interfere with the internal control (IC) of the virus marker in the sample. It is added to clinical samples and co-purified with clinical samples.
  • the internal control component is simultaneously detected via an IC Taqman probe labeled with a 705 nm fluorescent dye, which monitors the effects of the purification step and the PCR step.
  • the method of the present invention designs five sets of quantitative standards (QS) containing the full length of the HBV genome (pHBV-dimer), the full length of the HCV genome (pHCV-fl), and part of the HIV1 genome (5'LTR, pHIV1).
  • the plasmid of the inserted sequence, the partial HTLV1 genome (tat gene, pHTLV1-158), and the plasmid of the insertion sequence of the IC sequence, the signal of the sample and the quantitative standard are compared to calculate the number of viruses in the sample. .
  • the kit for simultaneously detecting and quantifying the multiple Taqman probe qPCR (M-tp-qPCR) of four blood-borne viruses of the present invention is 100% consistent with the results of other detection methods; and is measured by the kit of the present invention.
  • the viral copy number has a very high correlation with the standard, wherein the linear relationship (R 2 ) of HCV, HBV, HIV1, and HTLV1 is 0.9732, 0.9793, 0.9447, and 0.9788, respectively.
  • the kit of the invention can simultaneously detect four blood-borne viruses of HCV, HBV, HIV1, and HTLV1 in a single blood sample tube, or can be used for detecting a single blood-borne virus, which are highly specific (including Wide range of genotypic assays) and high sensitivity (10 to 100 copies/mL blood samples).
  • the kit for simultaneous detection and quantification of multiple blood-derived viruses with multiple Taqman probe qPCR can obtain viral quantitation results compared to the general detection methods that provide only qualitative results.
  • the kit of the present invention can be used for detecting high-throughput detection of 96-well plates within 1.5 hours, and the sealed test tube form can reduce the possibility of cross-contamination and has extremely high sensitivity and specificity. Therefore, the kit and method of the present invention can be used as blood screening for blood donors, detection of blood products, detection of cord blood stem cells, evaluation of the efficacy of anti-viral drugs, development of anti-viral drugs, and other uses.

Abstract

一种用于同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的试剂盒及方法。其中该试剂盒包含SEQ ID NO：1、SEQ ID NO：2、与SEQ ID NO：3所组成的组；SEQ ID NO：4、SEQ ID NO：5、与SEQ ID NO：6所组成的组；SEQ ID NO：7、SEQ ID NO：8、与SEQ ID NO：9所组成的组；以及SEQ ID NO：10、SEQ ID NO：11与SEQ ID NO：12所组成的组。目标检测的四种血源性病毒为乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、人类免疫缺陷病毒1型(HIV1)、以及人类T细胞淋巴瘤/白血病1型(HTLV1)。

Description

同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的试剂盒及方法 技术领域
本发明提供一种用于同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的试剂盒；特别是提供一种用于同时检测及定量分析乙型肝炎病毒(hepatitis B virus，HBV)、丙型肝炎病毒(hepatitis C virus，HCV)、人类免疫缺陷病毒1型(human immunodeficiency virus type1，HIV1)、以及人类T细胞淋巴瘤/白血病1型(human T-cell lymphoma/leukemia virus type 1，HTLV1)的血源性病毒的多重Taqman探针qPCR的试剂盒。
背景技术
血源性病毒(blood-borne viruses，BBV)主要经接触感染血液或血液制品、接触某些身体组织及体液、性接触及患病的母亲垂直传染给胎儿而传播。常见的血源性病毒包括乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、人类免疫缺陷病毒1型(HIV1)、人类T细胞淋巴瘤/白血病1型(HTLV1)、以及其他病毒。
HBV及HCV的感染是肝脏疾病中最常见及最严重的类型，全球有超过500万人感染这两种病毒。在台湾地区，有90％的成人曾经感染过HBV；约有15％至20％的人(约300万)是乙型肝炎病毒表面抗原(HBsAg)的病毒携带者。从1985年开始台湾实施全国性新生儿接种B型肝炎疫苗计划，以使乙型肝炎病毒表面抗原(HBsAg)的病毒携带者明显地减少。感染HBV的长期病症包含肝硬化及肝癌；由于肝炎及肝硬化而造成的死亡为所有死亡案例的第六名，而且在台湾地区，肝癌的死亡率仅次于肺癌，为第二个常见的癌症死亡原因。此外，在台湾地区约有30万人为HCV病毒携带者，大约是总人口的5％或HBV病毒携带者的1/10。
人类慢病毒亚科(lentivirinae)群组是由HIV-1以及HIV-2组合而成，其与严重免疫缺陷及获得性免疫缺陷综合征(Acquired lmmunodeficiency Syndrome，AIDS)有关。感染这些病毒的个体发病的可能性极高，感染HIV1的平均疾病症状发病时间约为10.7年，延后治疗或未接收到HIV检测结果或 延后进入HIV阳性反应照顾的患者是常见医疗卫生的问题。
人类致癌病毒(oncovirinae)包括HTLV-1及HTLV-2，近期称之为人类T-淋巴细胞病毒(human T-lymphotropic viruses)，其与良性及肿瘤淋巴增生性疾病、多种自身免疫疾病及免疫缺陷有关。然而，大多数个体感染这两种病毒仍可保持无症状的生活。由于输血而感染此病毒的可能性，美国食品及药物管理局(United States Food and Drug Administration)建议对所有血液捐赠者的血液除了进行HBV、HCV及HIV的筛选外，还针对HTLV1进行筛选。在美国，血液捐赠者HTLV1感染的血清阳性率在0.025％以内，在血清或血浆中以qRT-PCR定量HTLV1 RNA是最直接的检测病毒感染、复制及治疗效果的方法。
临床上对于上述四种血源性病毒分析的要求很严格。在2003年，台湾血库利用酶联免疫吸附测定(Enzyme-linked immunosorbent assay，ELISA)对30,000个血液样本进行筛选，其结果显示HBV的阳性率为1.09％、HCV的阳性率为0.28％、HIV1/2的阳性率为0.06％、HTLV的阳性率为0.05％，并进一步检测出梅毒螺旋体(Treponema pallidum，TP)的阳性率为0.07％。若使用核酸扩增技术(nucleic acid amplificationtechnology，NAT)，由于各种病毒的空窗期，核酸扩增技术的阳性率会比上述ELISA检测方法高。在美国地区，血液的需求量极大，在一天当中，约有38,000个人需要红细胞，其包含事故受害者、接受手术的患者以及接受白血病、癌症或其他例如镰状细胞病(sickle cell disease)、地中海贫血的疾病治疗的患者，因此每年有超过2千6百多万次的输血治疗。
台湾血库已利用酶联免疫吸附测定(ELISA)进行HBV、HCV、HIV1/2、以及HTLV1/2的筛选。HBV的检测是乙型肝炎病毒表面抗原，而其他病毒(HCV、HIV、以及HTLV)是进行相对应的抗原及抗体的检测。由于血液病毒具有各种空窗期，造成ELISA的检测有敏感度低、交叉反应性及假阴性的可能性的缺点。例如，HCV抗体的空窗期是70天、HIV抗体的空窗期是22天以及乙型肝炎病毒表面抗原的空窗期是56天。由于目前血清筛选测试无法检测出受感染的捐赠者的感染空窗期或无法检测出这些病毒的各种抗原种类，因此存在病原性病毒传播的风险。
核酸扩增技术(NAT)能够比前述筛选方法更早检测出病毒的存在，NAT已经使用作为血液捐赠者筛选的方法，其已证实其可用于检测出早期感染的有效性。先前研究已经表明，病毒核酸检测更能降低在空窗期间病原性病毒传播的风险。典型的NAT的步骤包括样本的制备、目标物的扩增、以及检测。在过去几年中，许多凝胶电泳、定性的单一目标PCR已经用于人类疱疹EB病毒(Epstein-Barr virus)、人类疱疹病毒、呼吸道病毒病原体、中枢神经系统病毒感染、虾病毒、以及6种食源性致病菌的检测。
目前，用于单一、二种、及三种血源性病毒的Taqman探针及信标(beacon)探针qPCR分析的类似方法已有报导。然而，上述方法皆有其方法上的缺点及不完整性，例如：三种血源性病毒皆用相同的荧光标记，无法得知阳性反应的样本是感染何种病毒；无法在三种血源性病毒上皆具有较高的专一性及敏感度以及无法获得定量的结果。
目前，分别检测四种血源性病毒(HCV、HBV、HIV、及HTLV)皆为大部分国家的血液筛检所要求。然而，现今在血液筛检方面缺少可应用在临床血液样本，于单一试管内便可同时检测及定量具有各种基因型的四种血源性病毒的含有引物组及探针组的试剂盒。
发明内容
本发明提供一种用于同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：1至SEQ ID NO：3所组成的组、SEQ ID NO：4至SEQ ID NO：6所组成的组、SEQ ID NO：7至SEQ ID NO：9所组成的组、以及SEQ ID NO：10至SEQ ID NO：12所组成的组；其中SEQ ID NO：1至SEQ ID NO：3用于检测乙型肝炎病毒；SEQ ID NO：4至SEQ ID NO：6用于检测丙型肝炎病毒；SEQ ID NO：7至SEQ ID NO：9用于检测人类免疫缺陷病毒1型；以及SEQ ID NO：10至SEQ ID NO：12用于检测人类T细胞淋巴瘤/白血病1型(HTLV1)，且该qPCR在单一反应混合物中进行。
本发明还提供一种用于检测及定量分析乙型肝炎病毒的Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：1至SEQ ID NO：3所组成的组。
本发明另提供一种用于检测及定量分析丙型肝炎病毒的Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：4至SEQ ID NO：6所组成的组。
本发明另提供一种用于检测及定量分析人类免疫缺陷病毒1型的Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：7至SEQ ID NO：9所组成的组。
本发明另提供一种用于检测及定量分析人类T细胞淋巴瘤/白血病1型(human T-cell lymphoma/leukemia virus type 1，HTLV1)的Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：10至SEQ ID NO：12所组成的组。
本发明还提供一种用于同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的方法，其包括：(a)提供生物样本；(b)提供引物组，包含SEQ ID NO：1与SEQ ID NO：2所组成的组；SEQ ID NO：4与SEQ ID NO：5所组成的组；SEQ ID NO：7与SEQ ID NO：8所组成的组；以及SEQ ID NO：10与SEQ ID NO：11所组成的组；(c)提供探针组，包括SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：9、以及SEQ ID NO：12；(d)使用该引物组、该探针组以及该生物样本进行qPCR；以及(e)观察生成的反应产物，以判断该血源性病毒是否存在于该生物样本中以及定量该血源性病毒，其中，SEQ ID NO：1至SEQ ID NO：3用于检测乙型肝炎病毒(HBV)；SEQ ID NO：4至SEQ ID NO：6用于检测丙型肝炎病毒(HCV)；SEQ ID NO：7至SEQ ID NO：9用于检测人类免疫缺陷病毒1型(HIV1)；以及SEQ ID NO：10至SEQ ID NO：12用于检测人类T细胞淋巴瘤/白血病1型(HTLV1)，且该qPCR在单一反应混合物中进行。
本发明的一个实施例中，其中SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：9、以及SEQ ID NO：12为5’端具有荧光染料且3’端具有荧光抑制分子的探针。
本发明的一个实施例中，其中SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：10、以及SEQ ID NO：11为引物。
有鉴于此，本发明提供一种在临床单一血液样本试管内同时检测及定量四种血源性病毒的各种基因型的试剂盒，其中该血源性病毒包括乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、人类免疫缺陷病毒1型(HIV1)、以及人类T细胞淋巴瘤/白血病1型(HTLV1)。本发明的包含引物及探针的试剂盒可用于在1.5个小时内检测96-孔板的高通量的检测方式，密封式试管形式可减少交叉污染的可能性，并具有极高的灵敏度及专一性。因此，本发明的试剂 盒可用于血液捐赠者的血液筛选、血液产品的检测、脐带血干细胞的检测、抗-病毒药物疗效的评估、抗-病毒药物的开发以及其它用途。
以下将配合附图进一步说明本发明的实施方式，下述所列举的实施例用以阐明本发明，并非用以限定本发明的范围，任何熟习此技术者，在不脱离本发明的精神和范围的情况下，当可做些许更动与润饰，因此本发明的保护范围当以随后所附的权利要求的范围为准。
附图说明
图1A至图1E为本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的定量增殖放大结果与标准曲线。定量标准品(QS)拷贝数为103至107；图1A至图1D的上部图、以及图1E为HBV、HCV、HIV1、及HTLV1四种病毒及内部控制组件(IC)的扩增图形；图1A至图1D的下部图为HBV、HCV、HIV1、以及HTLV1四种病毒的标准曲线。
图2为本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的凝胶电泳图，黑色指针表示四种病毒的PCR特定产物；白色指针表示内部控制组件(IC)的PCR产物。
图3A至图3D为本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的定量临床病毒样本的拷贝数结果。虚线表示平均病毒拷贝数；上部的实线表示定量标准品(QS)最大值；下部的实线表示定量标准品(QS)最小值，(图3A)为HCV阳性反应的样本；(图3B)为HBV阳性反应的样本；(图3C)为HIV1阳性反应的样本；(图3D)HTLV1阳性反应的样本。
图4A至图4D为本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的再现性，(图4A)HCV、(图4B)HBV、(图4C)HIV1、以及(图4D)HTLV1。
图5A至图5D为本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的敏感度，(图5A)HCV、(图5B)HBV、(图5C)HIV1、以及(图5D)HTLV1。
图6A至图6D为本发明的同时检测及定量分析四种血源性病毒的多重 Taqman探针qPCR(M-tp-qPCR)的试剂盒的动态范围分析，(图6A)HCV、(图6B)HBV、(图6C)HIV1、以及(图6D)HTLV1。
图7A至图7D为利用本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒分析样本的拷贝数与标准品连续稀释的拷贝数的关系图，(图7A)HCV、(图7B)HBV、(图7C)HIV1、以及(图7D)HTLV1。
图8A至图8D为本发明的分别利用单一血源性病毒的引物组及Taqman探针组定量已知标准品拷贝数结果的标准曲线，(图8A)为HCV阳性反应的样本；(图8B)为HBV阳性反应的样本；(图8C)为HIV1阳性反应的样本；(图8D)HTLV1阳性反应的样本。
图9A至图9D为本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒与单一血源性病毒的引物组及Taqman探针的qPCR反应的比较图，(图9A)HCV、(图9B)HBV、(图9C)HIV1、以及(图9D)HTLV1。
图10表示本发明的HBV引物核苷酸改变对qPCR特异性的影响，3’-G(G：C)代表本发明的Taqman探针qPCR的试剂盒；3’-C(C×C)代表改变检测HBV病毒的正向引物序列(SEQ ID NO：1)的3’末端的单一核苷酸。
图11表示本发明的HBV探针核苷酸改变对qPCR特异性的影响，C(C：G)代表本发明的Taqman探针qPCR反应的试剂盒；A(A×G)代表改变检测HBV病毒探针序列(SEQ ID NO：3)的中间位置第12个碱基C的单一核苷酸为A、G(G×G)代表改变检测HBV病毒探针序列(SEQ ID NO：3)中间位置第12个碱基C的单一核苷酸为G；T(T×G)代表改变检测HBV病毒探针序列(SEQ ID NO：3)的中间位置第12个碱基C的单一核苷酸为T。
图12表示本发明的HCV引物核苷酸改变对qPCR特异性的影响，3’-C(C：G)代表本发明的Taqman探针qPCR的试剂盒；3’-A(A×C)代表改变检测HCV病毒的正向引物序列(SEQ ID NO：4)的3’末端的单一核苷酸。
图13表示本发明的HIV1引物核苷酸改变对qPCR特异性的影响，3’-T(T：A)代表本发明的Taqman探针qPCR的试剂盒；3’-G(G×A)代表改变检测HIV1病毒的正向引物序列(SEQ ID NO：7)的3’末端的单一核苷酸。
图14表示本发明的HTLV1引物核苷酸改变对qPCR特异性的影响，3’-G(G：C)代表本发明的Taqman探针qPCR的试剂盒；3’-A(A×C)代表改变检测HTLV1病毒的正向引物序列(SEQ ID NO：10)的3’末端的单一核苷酸。
具体实施方式
本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒可用于同时或分别检测及定量活体外四种血源性病毒，包括乙型肝炎病毒、丙型肝炎病毒、人类免疫缺陷病毒1型、以及人类T细胞淋巴瘤/白血病1型。本发明的试剂盒可用于血液捐赠者的血液筛选、血液产品的检测、脐带血干细胞的检测、抗-病毒药物疗效的评估、抗-病毒药物的开发以及其它用途。本发明的试剂盒具有极高的聚合酶链式反应(PCR)的灵敏度、定量分析准确度、Taqman探针杂交的专一性、在Taq DNA聚合酶5’核酸酶活性的水解性且具有广泛的检测范围。本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒适用于两个步骤的定量反转录PCR检测方法：第一步，以随机六聚体将RNA病毒(包括HCV、HIV、以及HTLV)反转录为cDNA；以及第二步，将经纯化的HBV DNA病毒样本一起进行多重Taqman探针qPCR。
为测定本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR反应(M-tp-qPCR)的试剂盒的专一性，本发明采用477个阳性临床样本，其中包括57个HCV PCR阳性反应、91个HBV PCR阳性反应、20个HIV1 PCR阳性反应以及19个HTLV1 PCR阳性反应的样本；该些样本经定量PCR、实时(real-time)SYBR green PCR、RNA印迹法(Northern blotting)以及DNA测序进行病毒检测及鉴定。本发明使用MagBead病毒DNA/RNA试剂盒(Ambergene公司，台湾)或硅酸盐化薄膜柱病毒DNA/RNA纯化试剂盒的(silicated membrane column for viral purification DNA/RNA Kit)(Ambergene公司，台湾)纯化出病毒DNA/RNA。经本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒能准确鉴定出四种血源性病毒且没有假阴性发生，本发明的试剂盒能快速且准确地在受感染的血液样本中检测及定量HBV、HCV、HIV、以及HTLV1四种病毒。
实施例1 材料与方法
1.1 PCR引物、探针设计及病毒株
本发明利用Primer Express软件(Applied Biosystems，美国)设计寡核苷酸引物(表1)，在组成后计算所有的解链温度(melting temperature，Tm)值及利用聚合酶链式反应测试预期的产物大小。采用的寡核苷酸引物及Taqman探针由百力生物科技有限公司(Purigo Biotechnology Co.，Ltd，台湾)、基龙米克斯生物科技股份有限公司(Genomics BioSci&Tech，台湾)以及Integrated DNA Technologies(美国)合成而获得。Taqman探针分别采用FAM/BHQ1(HCVtp-313R-FAM)、HEX/BHQ1(HBVtp-395R-HEX)、CAL荧光红610/BHQ2(HIV1papPEtp-610)、Quasar 670/BHQ2(HTLV1-73-tp-670)以及Quasar705/BHQ2(Qbeta tp-705)标记，每一配对为荧光染料及荧光抑制分子。BioRad iQ5实时热循环仪具有5组激发滤波器组与放射滤波器组组，以705nm滤波器组组取代原来的TAMRA滤波器组组(TAMRA filter set)(第4通道)(channel 4)，该新的组合具有最小重迭的激发/发射波长，5个荧光染色可同时使用。
设计所有的病毒特异性的qPCR引物及探针，并经过多次的测试、修饰及改善。经过美国全国生物技术信息中心(National Center for Biotechnology information，NCBI)比对后，该引物及探针序列符合以下病毒株：HBV A(编号X70185)、HBV B(编号D00331)、HBV C(编号X01587)、HBV D(编号X72702)、HBV E(编号X75664)、HBV F(编号X75663)、HBV G(编号AF405706)、HBV H(编号EF157291)、HCV 1a(编号M67463)、HCV 1b(编号AB016785)、HCV 2a(编号AB047639)、HCV 2b(编号AB030907)、HCV 3a(编号AF046866)、HCV 3b(编号D49374)、HCV 4a(编号D45193、Y11604)、HCV 5a(编号D50466、Y13184)、HCV 6a(编号D88469，AY859526)、HIV1(编号NC_001802)、以及HTLV1(编号NC_001436)。
本发明设计的所有的引物及Taqman探针可检测特定病毒且包含特定病毒大部分的基因型，4对引物及探针可检测HBV病毒的A至H基因型、HCV的1至6基因型、HIV1、及HTLV1。HCV目标基因为5’UTR、HBV目标基因为S基因、HIV1目标基因为5’LTR、以及HTLV1目标基因为Tax基因； 其相对应的产物大小分别为120bp、150bp、＜100bp、以及73bp。这些引物、探针、目标基因及相对应的产物的大小列于表1及表2中。
表1 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的试剂盒的引物/Taqman探针
Figure PCTCN2016096964-appb-000001
表2 本发明的多重Taqman探针qPCR的试剂盒的引物、Taqman探针、定量标准品(QS)、产物大小、及四种血源性病毒的目标基因
Figure PCTCN2016096964-appb-000002
备注：FAM、HEX、610、670、705为荧光染料。BHQ1及BHQ2为荧光抑制分子。
表3 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针 qPCR(M-tp-qPCR)的试剂盒的五种探针标记荧光染料及荧光抑制分子的设计
Figure PCTCN2016096964-appb-000003
1.2 HBV/HCV/HIV1/HTLV1拷贝数标准品及基因型面板
依据PeliCheck HBV/HCV/HIV面板，HBV DNA拷贝数标准品(A基因型，拷贝数从0.1至30,000)、HCV RNA拷贝数标准品(1、2、3基因型，拷贝数从0.14至38,000)、HIV1 RNA拷贝数标准品(B、C、E基因型，拷贝数从0.075至25,000)。HTLV1 RNA拷贝数标准品经PCR、RNA印迹法及测序确定，该拷贝数由实验室HTLV1 SYBR green qRT-PCR分析确认。
使用Advanced Biotechnologies(ABI)公司的HTLV1病毒DNA控制组(感染细胞DNA，MT2病毒株)、HTLV2病毒DNA控制组(感染细胞DNA，C3-44(Mo))、HIV1(感染细胞，IIIB病毒株)、HIV2(感染细胞，NIH-Z病毒株)作为整合HIV1及HTLV拷贝数标准。HBV DNA的A、B、C、D、E、及F基因型由Cliniqa公司的血清制备(HBV基因型面板)。HBV的G及H基因型的PCR模板依据数据库的基因型序列合成的单股DNA。
1.3 临床样本
本发明的阳性临床样本数为477个，其来源包含：(i)150个血液样本，先前用于HBV基因型研究，包含91个HBV PCR阳性反应的样本，该91个HBV PCR阳性反应样本含有HBV的各基因型面板(genotypes panel)；(ii)190个血液样本，先前用于HCV基因型研究，包含57个HCV PCR阳性反应的样本，该57个HCV PCR阳性反应样本含有HCV的各基因型面板；(iii)87个血液样本，来自临床脐带血干细胞病毒污染筛选，包含20个HIV1 PCR阳性反应的样本；以及(iv)50个血液样本，先前用于脐带血干细胞病毒污染筛选，包含19个HTLV1 PCR阳性反应的样本。这些样本经定量PCR、实时 (real-time)SYBR green PCR、RNA印迹法、以及DNA测序进行病毒检测及鉴定。
1.4 内部控制组件
使用表2的内部控制组件(IC)，其可以监控提取步骤及确认PCR失败的可能性。内部控制组件的模板特别选择为4个目标病毒的非同源性序列，并将内部控制组件加入至每个样本中一起进行核酸提取及检测PCR扩增的步骤。其可作为提取及进行PCR的步骤效果的评估指标。在提取步骤开始时，在与浓缩的血清样本(200μL)混合之前先将内部控制组件加入至裂解缓冲液中(200μL/每个)；最后，将经纯化的病毒DNA/RNA沉淀物溶解于25μL的不含RNA酶(RNase-free)以及不含DNA酶(DNase-free)的溶液中。
定量标准品(QS)的制备
定量标准品(QS)作为标准曲线的非感染性DNA(如表2所示)。IC Taqman探针含有705nm荧光染料，IC不需加入进行RNA/DNA提取步骤。其制备为将病毒基因组全长或特定基因的PCR产物进行纯化，然后克隆至pEZT载体(TA克隆载体，Ambergene公司，台湾)。含有特定插入序列的重组质粒DNA通过硅酸盐化薄膜柱(silicated membrane column)或DEAE-亲合柱层析(DEAE-affinity column chromatography)方法纯化，然后以OD260/280吸光值定量，以计算出重组质粒DNA的理想拷贝数。每个病毒定量标准品模板均通过qPCR确认其拷贝数。
本发明的五组定量标准品(QS)为分别含有HBV基因组全长(pHBV-二聚体)、HCV基因组全长(pHCV-f1)、部分HIV1基因组(5’LTR，pHIV1-PapPEQF/QR)的插入序列的质粒、部分HTLV1基因组(Tat基因，pHTLV1-158)、以及IC序列的插入序列的质粒。
1.6 样本的收集
本发明使用病毒DNA/RNA纯化试剂盒(Ambergene公司，台湾)纯化血清或血浆样本。血清可用血清分离管收集；白细胞用无菌的EDTA或柠檬酸盐血液收集管(citrate blood collection tube)收集，含有抗凝血药物的样本不适合进行样本纯化。收集血液后六小时内，必须将血液收集管在室温下以3,000rpm离心10分钟以收集血清或血浆；再将血清或血浆吸取至聚丙烯试 管内，以作为病毒DNA/RNA纯化使用。或者，血清或血浆可存放至-20℃冰箱中以供之后使用。
1.7 样本的运送
白细胞必须在收集后六小时内在2至20℃下运送；血清或血浆可在2至8℃下运送或冷冻运送。
1.8 病毒DNA/RNA纯化
本发明使用MagBead病毒DNA/RNA试剂盒(Ambergene公司，台湾)或硅酸盐化包膜柱病毒DNA/RNA纯化试剂盒(silicated membrane column for viral purification DNA/RNA Kit)(Ambergene公司，台湾)并依据该商品制造商的操作手册从0.2mL血清或血浆样本纯化出病毒DNA/RNA，最后将病毒DNA/RNA溶解于50μL不含RNA酶以及不含DNA酶的溶液中。病毒DNA/RNA更进一步利用异丙醇沉淀以及以0.3M NaOAc pH 5.2溶液浓缩至25μL。使用随机六聚体将5μL病毒DNA/RNA进行cDNA合成，每一个PCR分析使用1μL cDNA。
1.9 cDNA的合成
cDNA的合成的反应总体积为10μL，包含1μL经纯化的病毒DNA/RNA、1μg随机六聚体引物、0.5μL 10mM dNTPs、2μL 5×第一股缓冲液(5x cDNA1st strand synthesis buffer)(250mM Tris-HCl pH 8.3、375mM KCl、15mM MgCl2)、0.5μL 0.1M DTT、0.1至0.25μL RNase抑制剂(40单位/μL)以及0.1至0.25μL MMLV RT(200U/μL，Ambergene公司，台湾)。该反应液先在25℃下反应5分钟使六聚体引物结合至RNA模板上，第一股cDNA在30℃下反应10分钟、40℃下反应40分钟、以及在95℃下反应5分钟而合成。在理想的状态下，在样本中同时有超过一种RNA目标序列会进行反转录。将完成的cDNA产物放置于冰上以进行多重Taqman探针qPCR(M-tp-qPCR)或单一Taqman探针qPCR；或将cDNA产物储存于-20℃冰箱中以供之后使用。
实施例2 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)
本发明利用具有5个滤膜组(FAM-495(激发)/520(发射)、HEX-535(激发)/556(发射)、CAL荧光红610-590(激发)/610(发射)、Quasar 670-647(激 发)/667(发射)、Quasar 705-690(激发)/705(发射)BioRad iQ5实时热循环仪。本发明的多重Taqman探针qPCR的方法的反应总体积为20μL，包含20mM Tris-HCl pH 8.8、10mM KCl、3mM MgCl2、0.1％ Triton X-100、0.1mg/mL BSA、每个dNTP 0.25mM、每个引物0.4mM、每个Taqman探针0.25mM以及2单位的热启动Taq DNA聚合酶(加热至42℃以上1分钟进行活化)(Ambergene公司，台湾)。使用八连排反应管(8-well strips)或96孔PCR微量板(Bio-Rad公司，美国)进行实验。反应条件为：95℃反应1分钟，再以95℃，反应1秒，55℃，反应30秒以及72℃反应30秒为一个循环，并重复此循环40次结束反应。利用iCycler iQ5软件进行数据分析而获得扩增图形(RFU对循环数)、标准曲线(Ct对模板拷贝数的log值)。
本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的特异性
本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的方法在缓冲液中具有定量标准品(QS)质粒及cDNA(目标病毒)模板、多重引物及探针混合的条件下进行检测的结果，本发明以斜率、R2、RFU、PCR效率(E)、Y截距评估qPCR的有效性，其中PCR效率(E)以E＝10(-1/斜率)定义，线性的相关系数(corrlation coefficient)，R2为测量该数据是否适合模块或数据是否适配于直线，其可能因吸取器吸取时的精确度及分析的范围所影响。如果R2≤0.985表示该分析不是可信赖的结果。如果一个或多个点在输入模板的最小值从图形的线性区域偏移，可能代表该数值超过分析敏感度。如果PCR具有100％效率，PCR产物的产量会随每个循环而倍数增加且标准曲线的斜率是-3.33(100＝100％＝10(-1/-3.33)-1)；标准曲线的斜率介于-3.9与-3.0之间(80至110％效率)通常是可以接受的。Y截距可被使用作为监控分析敏感度的参数之一，Y值超过40则表示为较慢的反应及低的分析敏感度，Y值低于40则表示为较快的反应且通常由不正确的模板复制计算或非特异性PCR产物的背景值。较高的镁离子浓度(Mg+2)会增加引物杂交的结合力，但亦会增加非专一性产物的产生及引物-引物二聚体的形成。
结果如图1A至图1E及表4、表5所示，本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒对HBV、 HCV、HIV1、以及HTLV1四种病毒的特异性极佳，HBV、HCV、HIV1、以及HTLV1的PCR效率分别为96.9、95、96.9、及113.2，该分析的线性关系是从1.000至0.997，该反应斜率分别为-3.399、-3.448、-3.399、及-3.014、以及该敏感度是高达101至102个拷贝数。该结果证实本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒可用于检测cDNA，含不同的起始浓度，含有4种血源性病毒序列的DNA模板均适用于本发明的分析方法；多重Taqman探针qPCR(M-tp-qPCR)或单一Taqman探针qPCR的试剂盒分别以定量标准品103至107或102至107拷贝数进行。
本发明特别设计不会干扰样本中病毒的内部控制组件(internal control，IC)，并将其加入至临床样本中，与临床样本同时纯化。内部控制组件经标记705nm荧光染料的IC Taqman探针检测，其可监控纯化步骤及PCR步骤的效果。此外，通过本发明的多重Taqman探针qPCR(M-tp-qPCR)的方法分析确认每个含有DNA插入序列的控制组标准品(QS)的拷贝数，之后经凝胶电泳分析确认产物大小与预期的一致(表2及图2)。
表4 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的特异性
Figure PCTCN2016096964-appb-000004
表5 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的敏感性、再现性、与标准品的线性关系
Figure PCTCN2016096964-appb-000005
Figure PCTCN2016096964-appb-000006
2.2 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒在临床血液样本中的应用
本发明共采用477个临床血液样本，利用MagBead病毒DNA/RNA试剂盒(Ambergene公司，台湾)或柱病毒DNA/RNA试剂盒(Column Viral DNA/RNA Kit)(Ambergene公司，台湾)纯化出病毒DNA/RNA。并事先将样本经由定量PCR、实时SYBR green PCR、RNA印迹法、以及DNA测序进行病毒检测及鉴定。
利用本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒检测HBV、HCV、HIV1、以及HTLV1四种病毒的辨识度100％与上述先前检测技术一致。如表6所示，这些样本混合物包含各种基因型面板标准品(HBV A至H基因型、HCV 1至6基因型、HIV1、以及HTLV1)。本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的辨识度100％与先前检测技术一致表示其可用于检测四种病毒的上述所有基因型。
表6 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的专一性
Figure PCTCN2016096964-appb-000007
本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的试剂盒定量的结果进一步提供每个阳性反应病毒样本的拷贝数。如图3A至图3D所示，其中57个HCV PCR阳性反应的样本，拷贝数分布在102至 107拷贝数/mL的范围内，每毫升血液样本的平均拷贝数为104至105拷贝数/mL；91个HBV PCR阳性反应的样本，拷贝数分布在102至108拷贝数/mL的范围内，每毫升血液样本的平均拷贝数为105拷贝数/mL；20个HIV1 PCR阳性反应的样本，拷贝数分布在＜102至104拷贝数/mL的范围内，每毫升血液样本的平均拷贝数为103拷贝数/mL；19个HTLV1 PCR阳性反应的样本，拷贝数分布在＜102至104拷贝数/mL的范围内，每毫升血液样本的平均拷贝数为103拷贝数/mL。
2.3 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的再现性及敏感度
本发明利用含有DNA插入序列的控制组件质粒定量标准品(QS)的六个重复样本进行扩增以确认再现性。输入每一个拷贝数(从100至106)以获得Ct值、Ct值平均值且因而计算标准偏差(SD)。结果如图4A至图4D、以及表5所示，本发明经由HBV、HCV、HIV1、以及HTLV1模板输入100至106拷贝数(从100至106)而分别获得Ct值的标准偏差(SD)为0.21至2.50、0.19至2.1、0.27至2.62、以及0.26至2.52。
本发明利用含有DNA控制组质粒定量标准品(QS)的十个重复样本连续稀释进行扩增以确认敏感度(从100至106)，并计算阳性检测率。如图5A至图5D、以及表6所示，所有HBV、HCV、HIV1、以及HTLV1四种病毒的检测敏感度高达10至102拷贝数。
2.4 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的动态范围(dynamic range)分析
本发明从101至109分别连续稀释四个病毒控制组件质粒DNA以确认四种病毒的动态范围以及定量分析。如图6A至图6D以及表5所示，所有四种病毒从101至109有一个广泛的动态范围。所有四种病毒分析显示极佳的有效性(斜率从-3.095至-3.4133)、极佳的线性关系(R2从0.999至0.9996)以及极佳的扩增率(Y截距从38.031至39.433)。
2.5 利用本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒分析所得的拷贝数与标准品连续稀释的拷贝数的比较
本发明以三个重复的样本进行分析，利用本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒分析获得的拷贝数与标准品连续稀释的拷贝数。如图7A至图7D所示，经本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒分析所得的拷贝数与标准品连续稀释的拷贝数具有高度线性关系(R2)，其中HCV线性关系(R2)为0.9732(从101至105)、HBV线性关系(R2)为0.9793(从101至106)、HIV1线性关系(R2)为0.9447(从101至104)、HTLV1线性关系(R2)为0.9788(从101至104)；并且较高的拷贝数比较低的拷贝数显示更具有关联性及更高的再现性。
实施例3 本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)与单一血源性病毒的Taqman探针qPCR的比较
本发明分别利用单一血源性病毒的引物组及Taqman探针与本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒的检测结果进行比较。
单一血源性病毒的Taqman探针qPCR反应总体积为20μL，包含20mM Tris-HCl，pH 8.8、10mM KCl、3mM MgCl2、0.1％ Triton X-100、0.1mg/mL BSA、每个dNTP 0.25mM、每个引物0.4mM、每个Taqman探针0.25mM、以及2单位的热启动Taq DNA聚合酶(加热至42℃以上1分钟进行活化)(Ambergene公司，台湾)。并使用八连排反应管(8-well strips)或96孔PCR微量板(Bio-Rad公司，美国)进行实验。反应条件为：95℃反应1分钟，再以95℃，反应1秒，55℃，反应30秒以及72℃反应30秒为一个循环，并重复此循环40次结束反应。其余方法均参照实施例2的检测方式。
本发明分别利用单一血源性病毒的引物组及Taqman探针定量已知标准品拷贝数结果的标准曲线，如图8A至图8D所示，其中57个HCV PCR阳性反应的样本，拷贝数分布在10至106拷贝数/mL的范围内，每毫升血液样本的平均拷贝数为1.36×105拷贝数/mL；91个HBV PCR阳性反应的样本，拷贝数分布在102至107拷贝数/mL的范围内，每毫升血液样本的平均拷贝数为4.02×106拷贝数/mL；20个HIV1 PCR阳性反应的样本，拷贝数分布在＜101至104拷贝数/mL的范围内，每毫升血液样本的平均拷贝数为1.89×104拷贝 数/mL；19个HTLV1 PCR阳性反应的样本，拷贝数分布在＜101至104拷贝数/mL的范围内，每毫升血液样本的平均拷贝数为3.46×104拷贝数/mL。
图9A至图9D显示单一血源性病毒的Taqman探针qPCR反应与同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的检测结果几乎一致，皆具有良好特异性。证实本发明的试剂盒除可同时检测及定量分析HCV、HBV、HIV1、以及HTLV1四种血源性毒外，亦可使用个别的引物组及探针检测个别病毒。
比较例1 改变本发明的引物或探针的核苷酸对PCR特异性的影响
为证实本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的引物及探针具有极佳的特异性，本发明通过改变本发明的引物或探针的核苷酸以确认本发明的引物或探针对qPCR反应的特异性。
C1.1 改变检测HBV病毒的正向引物序列SEQ ID NO：1的3’末端的核苷酸
本发明通过改变检测HBV病毒的正向引物序列(SEQ ID NO：1)的3’末端的单一核苷酸为SEQ ID NO：16，以确认本发明的引物对qPCR的特异性。结果如图10所示，该改变核苷酸的引物会改变动态范围而影响PCR的特异性，该改变核苷酸的引物的循环数(Ct)越高表示起始的拷贝数越少，证实本发明的用于检测及定量分析HBV的Taqman探针qPCR反应的试剂盒具有极高的灵敏度及专一性。
C1.2 改变检测HBV病毒探针序列(SEQ ID NO：3)的中间位置的单一核苷酸
本发明通过改变检测HBV病毒探针序列(SEQ ID NO：3)的中间位置第12个碱基C的单一核苷酸为A、G、或T，以确认本发明的探针对qPCR的特异性。结果如图11所示，通过本发明的HBV病毒探针序列(SEQ ID NO：3)的曲线证实本发明的HBV病毒探针序列完全匹配(match)于目标序列；其他三个单一核苷酸改变(第12个碱基由C改变为A、第12个碱基由C改变为G、以及第12个碱基由C改变为T)的探针则呈现负反应结果，证实本发明的用于检测及定量分析HBV的Taqman探针qPCR的试剂盒具有极高的灵 敏度及专一性。广泛来说，本发明的引物及探针只要改变一个碱基，灵敏度与特性便影响非常大，只以HBV病毒举例而已。
C1.3 改变检测HCV病毒的正向引物序列SEQ ID NO：4的3’末端的核苷酸
本发明通过改变检测HBV病毒的正向引物序列(SEQ ID NO：4)的3’末端的单一核苷酸C为A(SEQ ID NO：17)，以确认本发明的引物对qPCR的特异性。结果如图12所示，该改变核苷酸的引物会改变动态范围而影响PCR的特异性，该改变核苷酸的引物的循环数(Ct)越高表示起始的拷贝数越少，证实本发明的用于检测及定量分析HCV的Taqman探针qPCR的试剂盒具有极高的灵敏度及专一性。
C1.4 改变检测HIV1病毒的正向引物序列SEQ ID NO：7的3’末端的核苷酸
本发明通过改变检测HIV1病毒的正向引物序列(SEQ ID NO：7)的3’末端的单一核苷酸T为G(SEQ ID NO：18)，以确认本发明的引物对qPCR的特异性。结果如图13所示，该改变核苷酸的引物会改变动态范围而影响PCR的特异性，该改变核苷酸的引物的循环数(Ct)越高表示起始的拷贝数越少，证实本发明的用于检测及定量分析HCV的Taqman探针qPCR反应的试剂盒具有极高的灵敏度及专一性。
C1.5 改变检测HTLV1病毒的正向引物序列SEQ ID NO：10的3’末端的核苷酸
本发明通过改变检测HTLV1病毒的正向引物序列(SEQ ID NO：10)的3’末端的单一核苷酸G为A(SEQ ID NO：19)，以确认本发明的引物对qPCR的特异性。结果如图14所示，该改变核苷酸的引物会改变动态范围而影响PCR的特异性，该改变核苷酸的引物的循环数(Ct)越高表示起始的拷贝数越少，证实本发明的用于检测及定量分析HCV的Taqman探针qPCR的试剂盒具有极高的灵敏度及专一性。
综上所述，本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒检测HCV、HBV、HIV1、及HTLV1四种病毒相比于传统利用酶联免疫吸附测定更能有效的减少检测时间，同时对于 ELISA无法检测出带有突变病毒株及处于无症状空窗期的患者，本发明的方法亦能有效的被检测出来。本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒用于检测所有HBV基因型(A至H)、所有HCV基因型(1至6)、HIV1、及HTLV1。本发明未包含HIV2、及HTLV2的原因为这两个基因发生的机率较低对人类少有致病性。
本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的方法特别设计不会干扰样本中的病毒标记的内部控制组件(internal control，IC)，其加入至临床样本中，与临床样本共纯化。内部控制组件同时经由标记705nm荧光染料的IC Taqman探针检测，其可监控纯化步骤及PCR步骤的效果。此外，本发明的方法设计五组定量标准品(QS)，其分别含有HBV基因组全长(pHBV-二聚体)、HCV基因组全长(pHCV-fl)、部分HIV1基因组(5’LTR，pHIV1-PapPEQF/QR)的插入序列的质粒、部分HTLV1基因组(tat基因，pHTLV1-158)、以及IC序列的插入序列的质粒，将样本及定量标准品的信号比较后可计算出样本的病毒的数量。
本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒与其他检测方法的结果100％一致；且由本发明的试剂盒所测得的病毒拷贝数与标准品具有极高相关性，其中HCV、HBV、HIV1、及HTLV1的线性关系(R2)分别为0.9732、0.9793、0.9447、以及0.9788。本发明的试剂盒可在单一血液样本试管内同时检测HCV、HBV、HIV1、及HTLV1四种血源性病毒，或使用于检测单一血源性病毒，其皆具有极高的专一性(包含广泛的基因型检测范围)以及高度敏感度(10至100拷贝数/mL血液样本)。相比于一般检测方法仅提供定性的结果，本发明的同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR(M-tp-qPCR)的试剂盒可获得病毒定量的结果。此外，本发明的试剂盒可在1.5个小时内用于检测96-孔板的高通量检测方式，密封式试管形式可减少交叉污染的可能性，并具有极高的灵敏度及专一性。因此，本发明的试剂盒及方法可作为血液捐赠者的血液筛选、血液产品的检测、脐带血干细胞的检测、抗-病毒药物疗效的评估、抗-病毒药物的开发以及其它用途。

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  1. 一种用于同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：1至SEQ ID NO：3所组成的组、SEQ ID NO：4至SEQ ID NO：6所组成的组、SEQ ID NO：7至SEQ ID NO：9所组成的组、以及SEQ ID NO：10至SEQ ID NO：12所组成的组；
    其中SEQ ID NO：1至SEQ ID NO：3用于检测乙型肝炎病毒(HBV)；SEQ ID NO：4至SEQ ID NO：6用于检测丙型肝炎病毒(HCV)；SEQ ID NO：7至SEQ ID NO：9用于检测人类免疫缺陷病毒1型(HIV1)；以及SEQ ID NO：10至SEQ ID NO：12用于检测人类T细胞淋巴瘤/白血病1型(HTLV1)，
    且该qPCR在单一反应混合物中进行。
  2. 如权利要求1所述的试剂盒，其中SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：9、以及SEQ ID NO：12为5’端具有荧光染料且3’端具有荧光抑制分子的探针。
  3. 如权利要求1所述的试剂盒，其中SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：10、以及SEQ ID NO：11为引物。
  4. 一种用于检测及定量分析乙型肝炎病毒(HBV)的Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：1至SEQ ID NO：3所组成的组。
  5. 一种用于检测及定量分析丙型肝炎病毒(HCV)的Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：4至SEQ ID NO：6所组成的组。
  6. 一种用于检测及定量分析人类免疫缺陷病毒1型(HIV1)的Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：7至SEQ ID NO：9所组成的组。
  7. 一种用于检测及定量分析人类T细胞淋巴瘤/白血病1型(HTLV1)的Taqman探针qPCR的试剂盒，其包含：SEQ ID NO：10至SEQ ID NO：12所组成的组。
  8. 如权利要求4-7中任一项所述的试剂盒，其中SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：9、以及SEQ ID NO：12为5’端具有荧光染料且3’端具有荧光抑制分子的探针。
  9. 如权利要求4至7中任一项所述的试剂盒，其中SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：10、以及SEQ ID NO：11为引物。
  10. 一种用于同时检测及定量分析四种血源性病毒的多重Taqman探针qPCR的方法，其包括：(a)提供生物样本；(b)提供引物组，包括SEQ ID NO：1与SEQ ID NO：2所组成的组、SEQ ID NO：4与SEQ ID NO：5所组成的组、SEQ ID NO：7与SEQ ID NO：8所组成的组、以及SEQ ID NO：10与SEQ ID NO：11所组成的组；(c)提供探针组，包括SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：9、以及SEQ ID NO：12；(d)使用所述引物组、所述探针组以及所述生物样本进行qPCR；以及(e)观察生成的反应产物，以判断该血源性病毒是否存在于该生物样本中以及定量该血源性病毒，
    其中，SEQ ID NO：1至SEQ ID NO：3用于检测乙型肝炎病毒(HBV)；SEQ ID NO：4至SEQ ID NO：6用于检测丙型肝炎病毒(HCV)；SEQ ID NO：7至SEQ ID NO：9用于检测人类免疫缺陷病毒1型(HIV1)；以及SEQ ID NO：10至SEQ ID NO：12用于检测人类T细胞淋巴瘤/白血病1型(HTLV1)，且该qPCR在单一反应混合物中进行。
  11. 如权利要求10所述的方法，其中所述探针组为5’端具有荧光染料以及3’端具有荧光抑制分子的探针。
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CN111286557A (zh) * 2020-01-16 2020-06-16 山西国际旅行卫生保健中心(太原海关口岸门诊部) 一种检测输血性传播病原体的试剂组合、试剂盒及应用
CN113234866A (zh) * 2021-06-30 2021-08-10 上海君远生物科技有限公司 一种同步检测多项血液循环系统病原体的检测试剂盒及其检测方法
CN115948617A (zh) * 2022-12-23 2023-04-11 圣湘生物科技股份有限公司 检测血源性传播病毒的组合物、试剂盒、方法及其用途

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111926118A (zh) * 2020-08-21 2020-11-13 苏州育德扬生物技术有限公司 一种可同时检测艾滋病毒1、2型病毒以及人类t淋巴细胞白血病病毒1、2型的检测方法
CN114369649A (zh) * 2022-02-08 2022-04-19 山东见微生物科技有限公司 一种特异的选择扩增及多重pcr方法和应用
CN116694822B (zh) * 2023-06-16 2024-01-30 中山市中心血站 实时荧光环介导恒温扩增检测人类嗜t淋巴细胞i型病毒的方法及试剂盒

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1940087A (zh) * 2006-08-18 2007-04-04 上海科华生物工程股份有限公司 一种同步扩增检测肝炎及艾滋病毒核酸的方法及试剂盒
CN103184297A (zh) * 2011-12-27 2013-07-03 上海复星医学科技发展有限公司 一种荧光同步检测肝炎及艾滋病毒核酸的方法及试剂盒
CN105525035A (zh) * 2014-09-29 2016-04-27 天津华大基因科技有限公司 血液筛查试剂盒、方法及装置

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI507529B (zh) * 2011-10-28 2015-11-11 Taiwan Sugar Corp 同時檢測四種病毒之引子、引子對、方法及套組

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1940087A (zh) * 2006-08-18 2007-04-04 上海科华生物工程股份有限公司 一种同步扩增检测肝炎及艾滋病毒核酸的方法及试剂盒
CN103184297A (zh) * 2011-12-27 2013-07-03 上海复星医学科技发展有限公司 一种荧光同步检测肝炎及艾滋病毒核酸的方法及试剂盒
CN105525035A (zh) * 2014-09-29 2016-04-27 天津华大基因科技有限公司 血液筛查试剂盒、方法及装置

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CANDOTTI, D. ET AL.: "Multiplex Real-Time Quantitative RT - PCR Assay for Hepatitis B Virus, Hepatitis C Virus, and Human Immunodeficiency Virus Type 1", JOURNAL OF VIROLOGICAL METHODS, vol. 118, no. 1, 1 June 2004 (2004-06-01), pages 39 - 47, XP002548793 *
DEHEE, A. ET AL.: "Quantitation of HTLV-1 Proviral Load by a TaqMan Real-time PCR Assay", JOURNAL OF VIROLOGICAL METHODS, vol. 102, no. 1-2, 30 April 2002 (2002-04-30), pages 37 - 51, XP055426559 *
LI, GUIMIN: "Development and Application of Real-time PCR Assay for HBV, HCV and HIV-1 Nucleic Acids", MEDICINE & PUBLIC HEALTH, CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, vol. 6, 15 June 2012 (2012-06-15) *
Z IAEE, M. ET AL.: "The Prevalence of HTLV-1 and its Co-Infection with HCV, HBV and HIV in Hemophilic Patients", PAK. J. MED. SCI., vol. 31, no. 5, 31 October 2015 (2015-10-31), pages 1246 - 1249, XP055468098 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN111286557B (zh) * 2020-01-16 2023-12-08 山西国际旅行卫生保健中心(太原海关口岸门诊部) 一种检测输血性传播病原体的试剂组合、试剂盒及应用
CN111206123A (zh) * 2020-04-21 2020-05-29 珠海丽珠试剂股份有限公司 用于血液筛查的核酸组合物和试剂盒
CN113234866A (zh) * 2021-06-30 2021-08-10 上海君远生物科技有限公司 一种同步检测多项血液循环系统病原体的检测试剂盒及其检测方法
CN113234866B (zh) * 2021-06-30 2023-11-14 上海君远生物科技有限公司 一种同步检测多项血液循环系统病原体的检测试剂盒及其检测方法
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