TWI625395B - Kit for simultaneously detecting and quantifying four blood-borne viruses by a multiplex taqman probes quantitative pcr assay - Google Patents

Abstract

The present invention provides a kit for the simultaneous detection and quantification of multiple Taqman probe qPCR reactions for four blood-borne viruses. Wherein the kit comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, a primer probe set consisting of SEQ ID NO:9, and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12; four blood-borne viral lines of target detection For hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus type 1 (HIV1), and human T lymphoblastic virus type 1 (HTLV1), this primer probe combination and analytical method It has high sensitivity and specificity, and can be used as a blood donor's blood screening, blood product testing, cord blood stem cell testing, anti-viral drug efficacy evaluation, anti-viral drug development and other uses.

Description

Multiple Taqman probe qPCR reactions for simultaneous detection and quantification of four blood-borne viruses Set

The invention provides a kit for simultaneous detection and quantitative analysis of multiple Taqman probe qPCR reactions of four blood-borne viruses; in particular, 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 A set of multiple Taqman probe qPCR reactions for blood-borne viruses of HTLV1).

Blood-borne viruses (BBV) are mainly transmitted by the mother who is exposed to blood or blood products, exposure to certain body tissues and body fluids, sexual contact and disease, and the mother. Common blood-borne viruses include hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus type 1 (HIV1), human T Human T-cell lymphoma/leukemia virus 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. In Taiwan, 90% of the adult population has been infected with HBV; about 15% to 20% of the total population (about 3 million) is the carrier of hepatitis B virus surface antigen (HBsAg). Since 1985, Taiwan has implemented a national hepatitis B vaccination program for newborn vaccination to make the hepatitis B virus surface antigen (HBsAg) Significantly reduced. Long-term illnesses that infect HBV include cirrhosis and liver cancer; death from hepatitis and cirrhosis is the sixth in all deaths, and cancer death rate in Taiwan is second only to lung cancer, the second most common cancer cause of death. In addition, there are about 300,000 people in Taiwan who are HCV carriers, about 5% of the total population or 1/10 of the original HBV.

The human lentivirinae group is a combination of HIV-1 and HIV-2, which is associated with severe immunodeficiency and acquired lmmunodeficiency Syndrome (AIDS). Individuals infected with these viruses are highly likely to develop the disease. The average time to onset of HIV1 is about 10.7 years. Delayed treatment or failure to receive HIV test results or delay in HIV positive care is common to health care. problem.

Human oncogenic virus (oncovirinae) contains HTLV-1 and HTLV-2, recently known as human T-lymphotropic viruses, which are associated with benign and tumor lymphoproliferative disorders, various autoimmune diseases and immunodeficiencies related. However, most individuals who are infected with both viruses can remain asymptomatic. Due to the possibility of contracting the virus due to blood transfusions, the United States Food and Drug Administration recommends screening for HTLV1 in addition to screening for HBV, HCV and HIV in all blood donors. The seroprevalence of HTLV1 infection in blood donors in the United States is in the range of 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.

Clinically, the requirements for the analysis of the above four blood-borne viruses are very strict. In 2003, Taiwan blood bank used Enzyme-linked immunosorbent assay (ELISA) to screen 30,000 blood samples. The results showed that the positive rate of HBV was 1.09%, the positive rate of HCV was 0.28%, and HIV1/2. The positive rate was 0.06%, the positive rate of HTLV was 0.05%, and the positive probability of Treponema pallidum (TP) was 0.07%. If nucleic acid amplification technology (NAT) is used, the positive probability of nucleic acid amplification technology will be higher than the above ELISA detection method due to the empty window period of various viruses. In the United States, blood is in great demand. About 38,000 people need red blood cells in a 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 diseases of thalassemia.

Taiwan blood bank has used the enzyme immunoassay (ELISA) for screening HBV, HCV, HIV 1/2, and HTLV1/2. HBV detects hepatitis B virus surface antigen while others (HCV, HIV, and HTLV) perform detection of corresponding antigens and antibodies. Since blood viruses have various empty window periods, the detection of ELISA has the disadvantages of low sensitivity, cross-reactivity and the possibility of false negatives. For example, the empty window period of the HCV antibody is 70 days, the empty window period of the HIV antibody is 22 days, and 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 detect various antigen types of the virus, there is still a risk of transmission of the pathogenic virus.

Nucleic Acid Amplification (NAT) has the ability to detect the presence of a virus earlier than the aforementioned screening method, and NAT has been used as a method for blood donor screening, which has been shown to be useful for detecting early infection. Previous studies have shown that viral nucleic acid detection is more likely to reduce the risk of pathogenic viral transmission during empty windows. Typical NAT steps include preparation of samples, amplification of targets, and detection. In the past few years, many colloidal electrophoresis and qualitative single-target PCR have been used for human Epstein-Barr virus, human herpes virus, respiratory virus pathogen, central nervous system virus infection, Shrimp virus and detection of six foodborne pathogens.

At present, similar methods for Taqman probes and beacon probe qPCR analysis for single, two, and three blood-borne viruses have been reported; however, all of the above methods have their shortcomings and incompleteness, such as: All three blood-borne viruses were labeled with the same fluorescence, and it was not known which sample of the positive reaction was infected with the virus; it was not able to have high specificity and sensitivity on all three blood-borne viruses and could not obtain quantitative results.

Currently, the detection of four blood-borne viruses (HCV, HBV, HIV, and HTLV) is required by blood screening in most countries. However, in the current blood screening, there is a lack of a clinical blood sample that can simultaneously detect and quantify the four blood-borne viruses containing various genotypes containing primers and probe combinations in a single test tube.

The invention provides a method for simultaneously detecting and quantitatively analyzing four blood-borne viruses A kit of multiplex Taqman probe qPCR reactions comprising: SEQ ID NO: 1 to SEQ ID NO: 3, SEQ ID NO: 4 to SEQ ID NO: 6, 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 are for detecting the presence or absence of hepatitis B virus (HBV); SEQ ID NO: 4 to SEQ ID NO: 6 are for detecting the presence or absence of hepatitis C virus (HCV); SEQ ID NO: 7 to SEQ ID NO: 9 are for detecting human immunodeficiency virus Type 1 (human immunodeficiency virus type 1 , HIV1) is present or not; and SEQ ID NO: 10 to SEQ ID NO: 12 are used to detect human T-cell lymphoma/leukemia virus type 1 , HTLV1), and the instant polymerase chain reaction is carried out in a single reaction mixture.

The invention further provides a kit for detecting and quantifying a Taqman probe qPCR reaction of hepatitis B virus (HBV), comprising: SEQ ID NO: 1 to SEQ ID NO: Group.

The invention further provides a kit for detecting and quantifying a Taqman probe qPCR reaction of hepatitis C virus (HCV), comprising: SEQ ID NO: 4 to SEQ ID NO: Group.

The invention further provides a kit for detecting and quantifying a Taqman probe qPCR reaction of human immunodeficiency virus type 1 (HIV1), comprising: SEQ ID NO: 7 to SEQ ID NO: A group of nine.

The invention further provides a kit for detecting and quantifying a Taqman probe qPCR reaction of human T-cell lymphoma/leukemia virus type 1 (HTLV1), comprising: SEQ ID A group consisting of NO: 10 to SEQ ID NO: 12.

The invention also provides a method for simultaneously detecting and quantifying multiple Taqman probe qPCR reactions of four blood-borne viruses, comprising: (a) providing a biological sample; (b) providing a primer combination comprising SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8, and SEQ ID NO:10 and SEQ ID NO:11 a group of probes comprising: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12; (d) using the primer combination, the probe Combining the biological sample with a real-time polymerase chain reaction; and (e) observing the generated reaction product to determine whether the blood-borne virus is present in the biological sample and quantifying the blood source Viral virus, wherein step (e) SEQ ID NO: 1 to SEQ ID NO: 3 is for detecting the presence or absence of hepatitis B virus (HBV); SEQ ID NO: 4 to SEQ ID NO: 6 are for detecting The presence or absence of hepatitis C virus (HCV); SEQ ID NO: 7 to SEQ ID NO: 9 for detecting the presence or absence of human immunodeficiency virus type 1 (HIV1); and SEQ ID NO: 10 to SEQ ID NO : 12 is used to detect human T lymphoblastic virus type 1 (HTLV1), and the real-time polymerase chain reaction is carried out in a single reaction mixture.

In one embodiment of the invention, wherein SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, or SEQ ID NO: 12 has a fluorescent dye at the 5' end and a fluorescent light at the 3' end A probe that inhibits molecules.

In one embodiment of the invention, wherein 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 Or SEQ ID NO: 11 is a primer.

In view of the above, the present invention provides a kit for detecting and quantifying various genotypes of four blood-borne viruses simultaneously in a clinical single blood sample tube, wherein the blood-borne virus comprises hepatitis B virus (HBV), C Hepatitis B virus (HCV), human immunodeficiency virus type 1 (HIV1), and human T lymphoblastic virus type 1 (HTLV1). The set of primers and probes of the present invention can be used to detect the high-through throughput of 96-well plates within 1.5 hours. The sealed tube type can reduce the possibility of cross-contamination and has a pole. High sensitivity and specificity. Thus, the kit of the present invention can be used as a blood donor for blood screening, blood product testing, cord blood stem cell testing, evaluation of antiviral drug efficacy, development of antiviral drugs, and other uses.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

The first panels A to E are quantitative proliferative amplification results and standard curves of a set of multiple Taqman probe qPCR reactions (M-tp-qPCR) for simultaneous detection and quantification of four blood-borne viruses of the present invention. The quantitative standard (QS) copy number is 10 ³ to 10 ⁷ ; A to D above, and E is HBV, HCV, HIV1, and HTLV1 viruses and internal control (IC) expansion Increased graph; A to D The following figure shows the standard curves of four viruses: HBV, HCV, HIV1, and HTLV1.

The second figure is a colloidal electropherogram of a set of multiple Taqman probe qPCR reactions (M-tp-qPCR) for simultaneous detection and quantification of four blood-borne viruses of the present invention, and the black indicator indicates PCR specificity of four viruses. Product; white indicator indicates the PCR product of the internal control group (IC).

The third panels A to D are the copy number results of the quantitative clinical virus samples of the kit for simultaneous detection and quantitative analysis of multiple blood-borne viruses of multiple Taqman probe qPCR reactions (M-tp-qPCR). The dotted line indicates the average virus copy number; the upper solid line indicates the quantitative standard (QS) maximum; the lower solid line indicates the quantitative standard (QS) minimum value, (A) is the HCV positive reaction sample; (B) the HBV positive reaction (C) a sample that is HIV1 positive; (D) a sample of HTLV1 positive.

The fourth panels A to D are the reproducibility of the kit for simultaneous detection and quantification of multiple blood-borne viruses for multiple Taqman probe qPCR reactions (M-tp-qPCR), (A) HCV, (B) HBV, (C) HIV1, and (D) HTLV1.

The fifth panels A to D are the sensitivity of the kit for simultaneous detection and quantification of multiple Taqman probe qPCR reactions (M-tp-qPCR) of four blood-borne viruses, (A) HCV, (B) HBV, (C) HIV1, and (D) HTLV1.

6A to D are dynamic range analysis of a set of multiple Taqman probe qPCR reactions (M-tp-qPCR) for simultaneous detection and quantification of four blood-borne viruses of the present invention, (A) HCV, (B) HBV, (C) HIV1, and (D) HTLV1.

7A to D are sets of duplicate Taqman probe qPCR reactions (M-tp-qPCR) for simultaneous detection and quantification of four blood-borne viruses of the present invention, and the copy number of the sample is serially diluted with the standard. Copy number relationship diagram, (A) HCV, (B) HBV, (C) HIV1, and (D) HTLV1.

8A to D are standard curves of the results of quantification of known standards by using a single blood-borne virus primer group and a Taqman probe, respectively, (A) is a sample of HCV-positive reaction; (B) A sample that is positive for HBV; (C) a sample that is positive for HIV1; (D) a sample that is positive for HTLV1.

The ninth panels A to D are the primers for the simultaneous detection and quantification of the multiple Taqman probe qPCR reaction (M-tp-qPCR) of four blood-borne viruses and the introduction of a single blood-borne virus and Taqman. Comparison of probe qPCR reactions, (A) HCV, (B) HBV, (C) HIV1, and (D) HTLV1.

The tenth graph shows the effect of the nucleotide variation of the HBV primer of the present invention on the qPCR specificity, and the 3'-G (G:C) represents the kit of the Taqman probe qPCR reaction of the present invention; 3'-C (CxC) Represents a single nucleotide that changes the 3' end of the forward primer sequence (SEQ ID NO: 1) of the HBV virus.

The eleventh figure shows the effect of the nucleotide variation of the HBV probe of the present invention on qPCR specificity, C(C:G) represents the kit of the Taqman probe qPCR reaction of the present invention; A(AxG) table changes the detection of HBV The single nucleotide of the 12th base C of the intermediate position of the viral probe sequence (SEQ ID NO: 3) is A, G (GxG) represents the intermediate position of the detection HBV viral probe sequence (SEQ ID NO: 3). A single nucleotide of 12 bases C is G; T(TxG) represents a single nucleotide that changes the 12th base C of the intermediate position in which the HBV viral probe sequence (SEQ ID NO: 3) is detected.

The twelfth graph shows the effect of nucleotide variation of the HCV primer of the present invention on qPCR specificity, and 3'-C (C:G) represents a kit of Taqman probe qPCR reaction of the present invention; 3'-A (AxC) ) represents a single nucleotide that changes the 3' end of the forward primer sequence (SEQ ID NO: 4) that detects the HCV virus.

The thirteenth graph shows the effect of the nucleotide change of the HIV1 primer of the present invention on the qPCR specificity, and the 3'-T (T:A) represents the kit of the Taqman probe qPCR reaction of the present invention; 3'-G (GxA) ) represents a single nucleotide that changes the 3' end of the forward primer sequence (SEQ ID NO: 7) that detects the HIV1 virus.

Figure 14 is a graph showing the specificity of the nucleotide variation of the HTLV1 primer of the present invention on qPCR. 3'-G(G:C) represents a set of Taqman probe qPCR reactions of the present invention; 3'-A (AxC) represents a change of the positive primer sequence (SEQ ID NO: 10) for detecting HTLV1 virus 3 'Single nucleotide at the end.

The combination of multiple Taqman probe qPCR reaction (M-tp-qPCR) for simultaneous detection and quantification of four blood-borne viruses of the present invention can be used for simultaneous or separate detection and quantification of four blood-derived in vitro Blood borne viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus type 1 (human immunodeficiency virus type 1), and human hobby Human T-cell lymphoma/leukemia virus type 1 (human T-cell lymphoma/leukemia virus 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 present invention has extremely high sensitivity of polymerase chain reaction (PCR), quantitative analysis accuracy, specificity of Taqman probe hybridization, and hydrolyzability of Taq DNA polymerase 5' nuclease activity. And has a wide range of detection. The kit for simultaneously detecting and quantifying the multiple Taqman probe qPCR reaction (M-tp-qPCR) of four blood-borne viruses is suitable for the two-step quantitative reverse transcription PCR detection method: the first step, random six The hexameric hexamer reverse transcribes the RNA virus (including HCV, HIV, and HTLV) into cDNA; and the second step, the purified HBV DNA virus sample is subjected to a multiplex Taqman probe qPCR reaction.

To determine the specificity of the kit for simultaneous detection and quantification of multiple blood-borne viruses for multiple Taqman probe qPCR reactions (M-tp-qPCR), the present invention employs 477 positive clinical samples, including 57 HCV PCR positive reaction, 91 HBV PCR positive reactions, 20 HIV1 PCR positive reactions and 19 HTLV1 PCR positive samples; these samples were subjected to quantitative PCR, real-time SYBR green PCR, northern blotting and DNA sequencing for virus detection and identification. The present invention is purified using a MagBead virus DNA/RNA reagent set (Ambergene, Taiwan) or a silicated membrane column for viral purification DNA/RNA Kit (Ambergene, Taiwan). Viral DNA/RNA. Simultaneous detection and detection by the present invention Quantitative analysis of four blood-borne viruses with multiple Taqman probe qPCR reactions (M-tp-qPCR) can accurately identify four blood-borne viruses without pseudo-negative, the kit of the present invention can be fast and accurate The four viruses of HBV, HCV, HIV, and HTLV1 are detected and quantified in infected blood samples.

Example 1 Materials and Methods 1.1 PCR primer, probe design and virus strain

The present invention utilizes Primer Express software (Applied Biosystems, USA) to design oligonucleotide primers (Table 1), and calculates all melting temperature (Tm) values and polymerase chain reaction (polymerase chain reaction) after composition. PCR) tested the expected product size. The oligonucleotide primers and Taqman probes are composed of Purigo Biotechnology Co., Ltd., Taiwan, Genomics BioSci & Tech, Taiwan, and Integrated. DNA Technologies (USA) obtained by synthesis. The Taqman probes were FAM/BHQ1 (HCVtp-313R-FAM), HEX/BHQ1 (HBVtp-395R-HEX), CAL Fluorescent Red 610/BHQ2 (HIV1papPEtp-610), and Quasar 670/BHQ2 (HTLV1-73-, respectively). Tp-670) and Quasar 705/BHQ2 (Qbeta tp-705) markers, each paired with a fluorescent dye and a fluorescent inhibitor. The BioRad iQ5 real-time thermal cycler has five sets of excitation filters and radiation filters, replacing the original TAMRA filter set (channel 4) with a 705nm filter set. The new combination has a minimum overlap of excitation emission wavelengths and 5 fluorescent stains can be used simultaneously.

All virus-specific qPCR primers and probes were designed and tested, modified and improved. After alignment with the National Center for Biotechnology Information (NCBI), the primer and probe sequences conform to the following strains: 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. AB030907), HCV 3a (No. AF046866), HCV 3b (No. D49374), HCV 4a (No. D45193, Y11604), HCV 5a (No. D50466, Y13184), HCV 6a (No. D88469, AY859526), HIV1 (No. NC_001802), and HTLV1 (No. NC_001436).

All the primers and Taqman probes designed by the present invention can detect a specific virus and contain most of the genotypes of a specific virus, and 4 pairs of primers and probes can detect the A to H genotype of HBV virus, the 1 to 6 genotype of HCV, HIV1, and HTLV1. The HCV target gene is 5'UTR, the HBV target gene is S gene, the HIV1 target gene is 5'LTR, and the HTLV1 target gene is Tax gene; the corresponding product sizes are 120 bp, 150 bp, <100 bp, and 73 bp, respectively. The primers, probes, target genes and corresponding product sizes are shown in Tables 1 and 2.

Remarks: FAM, HEX, 610, 670, 705 are fluorescent dyes. BHQ1 and BHQ2 are fluorescent inhibitory molecules.

1.2 HBV/HCV/HIV1/HTLV1 copy number standard and genotype system group

According to the PeliCheck HBV/HCV/HIV system group, HBV DNA copy number standards (A genotype, copy number from 0.1 to 30,000), HCV RNA copy number standards (1, 2, 3 genotype, copy number from 0.14 to 38,000) ), HIV1 RNA copy number standards (B, C, E genotypes, copy number from 0.075 to 25,000). The HTLV1 RNA copy number standard was determined by PCR, Northern blotting, and sequencing, and the copy number was confirmed by laboratory HTLV1 SYBR green qRT-PCR analysis.

Use Advanced Biotechnologies (ABI)'s HTLV1 virus DNA control group (infected cell DNA, MT2 virus strain), HTLV2 virus DNA control group (infected cell DNA, C3-44 (Mo)), HIV1 (infected cells, IIIB virus strain), HIV2 (infected cells, NIH-Z virus) Strain) as an integrated HIV1 and HTLV copy number standard. The A, B, C, D, E, and F genotypes of HBV DNA were prepared from the serum of Cliniqa (HBV genotype system group). The PCR template for the G and H genotypes of HBV is a single-stranded DNA synthesized based on the genotype sequence of the library.

1.3 Clinical samples

The number of positive clinical samples of the present invention is 477, and the sources thereof include: (i) 150 blood samples previously used in the HBV genotype study, containing 91 HBV PCR positive reaction samples, and the 91 HBV PCR positive reaction samples contain HBV genotypes panel; (ii) 190 blood samples previously used in HCV genotyping studies, containing 57 HCV PCR positive samples containing HCV genotypes Systematic group; (iii) 87 blood samples from clinical cord blood stem cell virus contamination screening, containing 20 HIV1 PCR positive samples; and (iv) 50 blood samples previously used for cord blood stem cell virus contamination screening, A sample containing 19 HTLV1 PCR positive reactions. The samples were tested and identified by quantitative PCR, real-time SYBR green PCR, northern blotting, and DNA sequencing.

1.4 Internal Control Group

Use the internal control (IC) of Table 2, which monitors the extraction step and confirms the possibility of PCR failure. The template of the internal control group is specifically selected as a sequence of four non-homologous viruses of the target virus, and an internal control group is added to each sample for nucleic acid extraction and PCR amplification. It can be used as an indicator for evaluating the performance of steps in extraction and PCR. At the beginning of the extraction step, the internal control group was added to the lysis buffer (200 μL/each) prior to mixing with the concentrated serum sample (200 μL); finally, the purified viral DNA/RNA precipitate was Dissolved in 25 μL of RNase-free and DNase-free solution.

1.5 Preparation of Quantitative Standards (QS)

The quantitative standard (QS) is a non-infectious DNA as a standard curve (as shown in Table 2). The IC Taqman probe contains a 705 nm fluorescent dye and the IC does not need to be added for the RNA/DNA extraction step. It is prepared by purifying the full length of a viral gene or a PCR product of a specific gene, and then selecting it into a pEZT vector (TA selection vector, Ambergene, Taiwan). Recombinant plastid DNA containing a specific insert sequence is purified via a silicated membrane column or a DEAE-affinity column chromatography method, and then quantified by OD _260/280 absorbance. To calculate the ideal copy number of recombinant plastid DNA. Each viral quantitative standard template confirmed its copy number via qPCR.

The five sets of quantitative standards (QS) of the present invention contain 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-PapPEQF/). The plastid of the inserted sequence of QR), part of the HTLV1 gene (tat gene, pHTLV1-158), and the plastid of the insertion sequence of the IC sequence.

1.6 Collection of samples

The present invention uses a viral DNA/RNA purification reagent set (Ambergene, Taiwan) to purify serum or plasma samples. Serum can be collected in a serum separator tube; white blood cells are in sterile EDTA or citrate blood collection tubes, and samples containing anticoagulant drugs are not suitable for sample purification. Within six hours of collecting the blood, the blood collection coagulation tube must be centrifuged at 3,000 rpm for 10 minutes at room temperature to collect serum or plasma; the serum or plasma is then aspirated into a polypropylene tube for use as viral DNA/RNA purification. Alternatively, serum or plasma can be stored in a -20 ° C freezer for later use.

1.7 Sample transportation

White blood cells must be transported at 2 to 20 °C within six hours of collection; serum or plasma can be shipped or frozen at 2 to 8 °C.

1.8 Viral DNA/RNA purification

The present invention uses a MagBead virus DNA/RNA reagent set (Ambergene Corporation, Taiwan) or a silicated membrane column for viral purification DNA/RNA Kit (Ambergene Corporation, Taiwan) and The product manufacturer's operating manual purifies the virus from 0.2 mL serum or plasma samples. DNA/RNA, and finally the viral DNA/RNA was dissolved in 50 μL of RNase-free and DNase-free solution. The viral DNA/RNA was further precipitated with isopropanol and concentrated to 25 μL with a 0.3 M NaOAc pH 5.2 solution. 5 μL of viral DNA/RNA was subjected to cDNA synthesis using a random hexamer, and 1 μL of cDNA was used for each PCR analysis.

1.9 Synthesis of cDNA

cDNA synthesis reaction for a total volume of 10μL, containing 1μL purified viral DNA / RNA, 1μg of random hexamer primer, 0.5μL 10mM dNTPs, 2μL 5x first strand buffer ^{(5x cDNA 1 st strand synthesis buffer} ) (250mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl ₂ ), 0.5 μL 0.1 M DTT, 0.1 to 0.25 μL RNase inhibitor (40 units/μL), and 0.1 to 0.25 μL MMLV RT (200 U/μL, 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 cDNA was synthesized by reacting at 30 ° C for 10 minutes, 40 ° C for 40 minutes, and reacting at 95 ° C for 5 minutes. 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 reaction (M-tp-qPCR) or single Taqman probe qPCR reaction; or the cDNA product was stored in a -20 ° C refrigerator for later use.

Example 2 Multiple Taqman probe qPCR reaction (M-tp-qPCR) for simultaneous detection and quantification of four blood-borne viruses of the present invention

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 instant thermal cycler. The method of the multiple Taqman probe qPCR of the present invention has a total reaction volume of 20 μL, 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 hot start (hot start) Taq DNA polymerase (heated to 42 ° C for 1 minute for activation) (Ambergene, Taiwan) and use eight-well strips or 96-well PCR microplates (Bio-Rad , USA) The experiment was carried out under the following conditions: reaction at 95 ° C for 1 minute, reaction at 95 ° C, 1 second, 55 ° C, 30 seconds, and 72 ° C for 30 seconds, and repeating the reaction 40 times to end the reaction. Using iCycler The iQ5 software performs data analysis to obtain an amplification pattern (RFU vs. cycle number) and a standard curve (Ct vs. template copy number log value).

2.1 The specificity of the kit for simultaneous detection and quantification of multiple Taqman probe qPCR reactions (M-tp-qPCR) of four blood-borne viruses of the present invention

The method for simultaneously detecting and quantifying multiple Taqman probe qPCR reactions (M-tp-qPCR) of four blood-borne viruses in the present invention has a quantitative standard (QS) plastid and cDNA (target virus) template in a buffer As a result of the detection under the condition of mixing multiple primers and probes, the present invention evaluates the validity of qPCR by slope, R ² , RFU, PCR efficiency (E) and Y intercept, wherein the PCR efficiency (E) is E=10. ^(-1/slope) defines a linear correlation coefficient, and R ² is a measure of whether the data is suitable for the module or whether the data is adapted to a straight line, which may be due to the accuracy of the suction and the range of analysis. influences. If R ² ≦ 0.985 indicates that the analysis is not a reliable result. If one or more points are offset from the linear region of the graph at the minimum of the input template, it may represent that the value exceeds the analytical sensitivity. If the PCR is 100% efficient, the yield of the PCR product will increase with each cycle and the slope of the standard curve is -3.33 (100 = 100% = 10 ^(-1/-3.33) -1); the slope of the standard curve Between -3.9 and -3.0 (80 to 110% efficiency) is generally acceptable. 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. A higher magnesium ion concentration (Mg ⁺² ) increases the binding force of the primer hybridization, but also increases the production of non-specific products and the formation of primer-primer dimers.

Results As shown in the first panels A to E and Tables 4 and 5, the kit for simultaneous detection and quantification of multiple blood-borne viruses of multiple Taqman probe qPCR reactions (M-tp-qPCR) to HBV The HCV, HIV1, and HTLV1 viruses have excellent specificity. The PCR efficiencies of HBV, HCV, HIV1, and HTLV1 are 96.9, 95, 96.9, and 113.2, respectively. The linear relationship of the analysis is from 1.000 to 0.997. The reaction slopes were -3.499, -3.448, -3.49, and -3.014, respectively, and the sensitivity was as high as 10 ¹ to 10 ² copies. The results demonstrate that the kit for simultaneous detection and quantification of multiple blood-borne viruses of multiple Taqman probe qPCR reactions (M-tp-qPCR) of the present invention can be used to detect cDNA with different initial concentrations, including four blood sources. The DNA template of the viral sequence is suitable for the analysis method of the present invention; the set of multiple Taqman probe qPCR reaction (M-tp-qPCR) or single Taqman probe qPCR reaction is respectively used to quantify the standard 10 ³ to 10 ⁷ or 10 ² to 10 ⁷ copy numbers are performed.

The present invention is specifically designed to not interfere with the internal control group (IC) of the virus in the sample and to be added to the clinical sample for simultaneous purification with the clinical sample. The internal control group was 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. Furthermore, the copy number of each control group standard (QS) containing the DNA insert sequence was confirmed by the method of the multiplex Taqman probe qPCR reaction (M-tp-qPCR) of the present invention, and then the product size was confirmed by colloidal electrophoresis analysis. Expected agreement (Table 2 and Figure 2).

2.2 Simultaneous detection and quantification of a set of multiple Taqman probe qPCR reactions (M-tp-qPCR) of four blood-borne viruses in clinical blood samples

The invention uses a total of 477 clinical blood samples, using the MagBead virus DNA/RNA reagent set (Ambergene, Taiwan) or the silicated membrane column for viral purification DNA/RNA kit. (Ambergene, Taiwan) purified viral DNA/RNA, and the samples were tested and identified by quantitative PCR, real-time SYBR green PCR, northern blotting, and DNA sequencing.

Using the multi-Taqman probe qPCR reaction (M-tp-qPCR) kit for simultaneous detection and quantification of four blood-borne viruses of the present invention to detect the recognition of HBV, HCV, HIV1, and HTLV1 viruses by 100% and The above previous detection techniques are consistent. As shown in Table 6, the sample mixes included various genotype system group standards (HBV A to H genotype, HCV 1 to 6 genotype, HIV1, and HTLV1). The set of multi-Taqman probe qPCR reaction (M-tp-qPCR) for simultaneous detection and quantification of four blood-borne viruses of the present invention has a recognition degree of 100%, which is consistent with previous detection techniques, and can be used for detecting four viruses. All of the above genotypes.

The results of the kit 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. As shown in the third panel A to D, among the 57 samples positive for HCV PCR, the copy number is distributed in the range of 10 ² to 10 ⁷ copies/mL, and the average copy number per ml of blood sample is 10 ⁴ to 10 ⁵ copies/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 For HIV1 PCR-positive samples, the copy number was distributed in the range of <10 ² to 10 ⁴ copies/mL, and the average copy number per ml of blood sample was 10 ³ copies/mL; 19 samples of HTLV1 PCR positive reaction, The copy 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.

2.3 The reproducibility and sensitivity of the kit for simultaneous detection and quantification of multiple Taqman probe qPCR reactions (M-tp-qPCR) of four blood-borne viruses

The present invention utilizes six replicate samples of a control panel plastid 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). As a result of the fourth panels A to D, and shown in Table 5, the present invention is by HBV, HCV, HIV1, HTLV1 template and input ¹⁰⁰ to ¹⁰⁶ were given a standard number of copies of Ct values (from ¹⁰⁰ to ¹⁰⁶⁾ The difference (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 DNA control plastid quantification standards (QS) for amplification to confirm sensitivity (from 10 ⁰ to 10 ⁶ ) and calculate a positive detection rate. As shown in Figures 5A to D and Table 6, all HBV, HCV, HIV1, and HTLV1 viruses have detection sensitivities as high as 10 to 10 ² copies.

2.4 Dynamic range analysis of a set of multiple Taqman probe qPCR reactions (M-tp-qPCR) for simultaneous detection and quantification of four blood-borne viruses of the present invention

The present invention serially dilutes four viral control group plastid DNA from 10 ¹ to 10 ⁹ via the subject to confirm the dynamic range of the four viruses and quantitative analysis. As shown in Figures 6 through A 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) .

2.5 Comparison of the copy number obtained by the kit analysis of the multiple Taqman probe qPCR reaction (M-tp-qPCR) of the simultaneous detection and quantification of four blood-borne viruses of the present invention with the serial dilution of the standard product

The present invention uses three repeated samples for analysis. The copy number obtained by the kit analysis of the multiple Taqman probe qPCR reaction (M-tp-qPCR) for simultaneously detecting and quantifying the four blood-borne viruses of the present invention is continuous with the standard product. The diluted copy number. As shown in the seventh panel A to D, the copy number obtained by the kit analysis of the multiple Taqman probe qPCR reaction (M-tp-qPCR) for simultaneously detecting and quantifying the four blood-borne viruses of the present invention is continuous with the standard product. The diluted 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 ), and the HBV linear relationship (R ² ) is 0.9793 (from 10 ¹ to 10 ⁶ ), The HIV1 linear relationship (R ² ) was 0.9447 (from 10 ¹ to 10 ⁴ ), the HTLV1 linear relationship (R ² ) was 0.9788 (from 10 ¹ to 10 ⁴ ); and the higher copy number compared to the lower copy number showed a higher Relevance and high reproducibility.

Example 3 Comparison of qq Reaction of Multi-Taqman Probe qPCR Reaction (M-tp-qPCR) Simultaneously Detecting and Quantifying Analysis of Four Blood-borne Viruses with Taqman Probe of 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 of a set of multiple Taqman probe qPCR reactions (M-tp-qPCR) of four blood-borne viruses. The results were compared.

The total volume of the Taqman probe qPCR reaction of a single blood-borne virus is 20 μL, 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 unit 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). The reaction conditions were as follows: reaction at 95 ° C for 1 minute, and further reaction at 95 ° C, 1 second, 55 ° C, 30 seconds, and 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 the eighth panel A to D, wherein 57 samples of HCV PCR positive reaction, copy number Distributed 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 HBV PCR positive samples, copy number distribution is 10 ² to 10 ⁷ In the range of copy 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 between <10 ¹ and 10 ⁴ copies/mL Within the range, 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 The average copy number of the blood sample was 3.46 x 10 ⁴ copies/mL.

The ninth panels A to D show that the Taqman probe qPCR reaction of a single blood-borne virus simultaneously detects and quantifies the results of the multiple Taqman probe qPCR reaction (M-tp-qPCR) of four blood-borne viruses. , all have good specificity. It is confirmed that the kit of the present invention can simultaneously detect and quantitatively analyze four blood-borne viruses of HCV, HBV, HIV1, and HTLV1, and can also detect individual viruses by using individual primer groups and probes.

Comparative Example 1 Effect of changing the nucleotide specificity of the primer or probe of the present invention on PCR specificity

In order to confirm that the primers and probes of the multiple Taqman probe qPCR reaction (M-tp-qPCR) for simultaneously detecting and quantifying the four blood-borne viruses of the present invention have excellent specificity, the present invention changes the primer of the present invention. Or the nucleotide of the probe to confirm the specificity of the primer or probe of the present invention for the qPCR reaction.

C1.1 Altering the nucleotide of the 3' end of the forward primer sequence of SEQ ID NO: 1 for detection of HBV virus

The present invention recognizes that the single nucleotide G at the 3' end of the forward primer sequence (SEQ ID NO: 1) of the HBV virus is C (SEQ ID NO: 16) to confirm the specificity of the primer of the present invention for the qPCR reaction. Sex. As shown in the tenth figure, the change of the primer of the nucleotide changes the dynamic range and affects the specificity of the PCR, and the increase in the number of cycles (Ct) of the primer of the altered nucleotide indicates that the initial copy number is less. It was confirmed that the kit for detecting and quantifying the Taqman probe qPCR reaction of HBV of the present invention has extremely high sensitivity and specificity.

C1.2 Alteration of a single nucleotide detecting the intermediate position of the HBV viral probe sequence (SEQ ID NO: 3)

The present invention recognizes the present invention by changing the single nucleotide of the 12th base C of the intermediate position of the HBV virus probe sequence (SEQ ID NO: 3) to A, G, or T. The specificity of the probe for the qPCR reaction. Results As shown in Fig. 11, the HBV virus probe sequence of the present invention was completely matched to the target sequence via the curve of the HBV virus probe sequence (SEQ ID NO: 3) of the present invention; the other three single nuclei The change in the glycosidic acid (the 12th base changed from C to A, the 12th base changed from C to G, and the 12th base changed from C to T) showed a negative reaction result. It is confirmed that the kit for detecting and quantifying the Taqman probe qPCR reaction of HBV of the present invention has extremely high sensitivity and specificity. Broadly speaking, the primers and probes of the present invention have a very large influence on sensitivity and characteristics as long as they change by one base, and only the HBV virus is exemplified.

C1.3 Altering the nucleotide of the 3' end of the forward primer sequence of SEQ ID NO: 4 for detecting HCV virus

The present invention recognizes that the single nucleotide C at the 3' end of the forward primer sequence (SEQ ID NO: 4) of the HCV virus is A (SEQ ID NO: 17) to confirm the specificity of the primer of the present invention for the qPCR reaction. Sex. As shown in Fig. 12, the change of the primer of the nucleotide changes the dynamic range and affects the specificity of the PCR. The increase in the number of cycles (Ct) of the primer of the altered nucleotide indicates that the initial copy number is less. It is confirmed that the kit of the Taqman probe qPCR reaction for detecting and quantifying HCV of the present invention has extremely high sensitivity and specificity.

C1.4 Altering the nucleotide of the 3' end of the forward primer sequence of SEQ ID NO: 7 for detecting HIV1 virus

The present invention recognizes that the single nucleotide T at the 3' end of the forward primer sequence (SEQ ID NO: 7) of the HIV1 virus is G (SEQ ID NO: 18) to confirm the specificity of the primer of the present invention for the qPCR reaction. Sex. As shown in the thirteenth image, the change of the primer of the nucleotide changes the dynamic range and affects the specificity of the PCR. The increase in the number of cycles (Ct) of the primer of the altered nucleotide indicates that the initial copy number is less. It is confirmed that the kit for detecting and quantifying the Taqman probe qPCR reaction of HIV1 of the present invention has extremely high sensitivity and specificity.

C1.5 Alteration of the nucleotide of the 3' end of the forward primer sequence of SEQ ID NO: 10 for detecting the HTLV1 virus

The present invention recognizes that the single nucleotide G at the 3' end of the forward primer sequence (SEQ ID NO: 10) of the HTLV1 virus is A (SEQ ID NO: 19) to confirm the specificity of the primer of the present invention for the qPCR reaction. Sex. As shown in Fig. 14, the change of the primer of the nucleotide changes the dynamic range and affects the specificity of the PCR, and the number of cycles (Ct) of the primer of the altered nucleotide is increased. High indicates that the initial copy number is less, and the set of the Taqman probe qPCR reaction for detecting and quantifying HTLV1 of the present invention is confirmed to have extremely high sensitivity and specificity.

In summary, the multi-Taqman probe qPCR reaction (M-tp-qPCR) kit for simultaneous detection and quantification of four blood-borne viruses of the present invention detects HCV, HBV, HIV1, and HTLV1 viruses. It is more effective to reduce the detection time by using the Enzyme-linked immunosorbent assay (ELISA), and the method of the present invention is also unable to detect a patient with a mutant virus and being in an asymptomatic window during the ELISA. Can be detected effectively. The kit for simultaneous detection and quantification of multiple blood-borne viruses of multiple Taqman probe qPCR reactions (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 probability of occurrence of the two genes is less pathogenic to humans.

The method for simultaneously detecting and quantifying the multiple Taqman probe qPCR reaction (M-tp-qPCR) of four blood-borne viruses of the present invention is specifically designed to not interfere with the internal control group (IC) of virus calibration in the sample, and the addition thereof To clinical samples, co-purified with clinical samples. The internal control group was 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. In addition, the method of the present invention designs five sets of quantitative standards (QS), which respectively contain 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, The plastid of the inserted sequence of pHIV1-PapPEQF/QR), part of the HTLV1 gene (tax gene, pHTLV1-158), and the plastid of the insertion sequence of the IC sequence, the sample can be compared with the signal of the quantitative standard to calculate the sample. The number of viruses.

The set of multiple Taqman probe qPCR reactions (M-tp-qPCR) for simultaneous detection and quantification 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 a wide range of Genotype detection range) and high sensitivity (10 to 100 copies/mL blood sample). Compared with the general detection method, only qualitative results are provided, and the results of the present invention for simultaneous detection and quantification of multiple blood-borne viruses of multiple Taqman probe qPCR reactions (M-tp-qPCR) can obtain the results of virus quantification. In addition, the kit of the present invention can be used in a high-throughput assay for detecting 96-well discs within 1.5 hours. The sealed tube type reduces the possibility of cross-contamination and has extremely high sensitivity. Specificity. Thus, the kits and methods of the present invention can be used for blood screening by blood donors, testing 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.

<110> Xuji Technology Co., Ltd.

<120> Set of multiple Taqman probe qPCR reactions for simultaneous detection and quantification of blood-borne viruses

<130> 105B0129-I1

<160> 19

<170> PatentIn version 3.5

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<110> Xuji Technology Co., Ltd.

<120> Primer combination, kit and method for multiple Taqman probe qPCR reactions for simultaneous detection and quantification of blood-borne viruses

<130> 105B0129-I1

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Claims (8)

A kit for simultaneous detection and quantification of multiple Taqman probe qPCR reactions for four blood-borne viruses comprising: SEQ ID NO: 1 to SEQ ID NO: 3, SEQ ID NO: 4 to SEQ ID NO a group consisting of SEQ ID NO: 7 to SEQ ID NO: 9, and SEQ ID NO: 10 to SEQ ID NO: 12; wherein SEQ ID NO: 1 to SEQ ID NO: 3 are for detecting B Hepatitis B virus (HBV) is present; SEQ ID NO: 4 to SEQ ID NO: 6 are used to detect the presence or absence of hepatitis C virus (HCV); SEQ ID NO: 7 to SEQ ID NO: 9 is for detecting the presence or absence of human immunodeficiency virus type 1 (HIV1); and SEQ ID NO: 10 to SEQ ID NO: 12 are for detecting human T lymphoblastic virus The first type (human T-cell lymphoma/leukemia virus type 1, HTLV1), and the instant polymerase chain reaction is carried out in a single reaction mixture. The kit of claim 1, wherein 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 3 'The probe with a fluorescent inhibitory molecule at the end. The kit of claim 1, wherein 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. A kit for detecting and quantifying a Taqman probe qPCR reaction of hepatitis B virus (HBV), comprising: a group consisting of SEQ ID NO: 1 to SEQ ID NO: 3. The kit of claim 4, wherein SEQ ID NO: 3 is a probe having a fluorescent dye at the 5' end and a fluorescent inhibitory molecule at the 3' end. The kit of claim 4, wherein SEQ ID NO: 1, SEQ ID NO: 2 The system is an introduction. A method for simultaneously detecting and quantifying multiple Taqman probe qPCR reactions of four blood-borne viruses, comprising: (a) providing a biological sample; (b) providing a primer combination comprising SEQ ID NO: 1 And the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 10 and SEQ ID NO: 11; c) providing a probe combination comprising SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12; (d) using the primer combination, the probe combination, and the biological sample Performing a real-time polymerase chain reaction; 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 Step (e) SEQ ID NO: 1 to SEQ ID NO: 3 are for detecting the presence or absence of hepatitis B virus (HBV); SEQ ID NO: 4 to SEQ ID NO: 6 are for detecting hepatitis C virus ( HCV) presence or absence; SEQ ID NO: 7 to SEQ ID NO: 9 for detecting the presence or absence of human immunodeficiency virus type 1 (HIV1); and SEQ ID NO: 10 To SEQ ID NO: 12 is used to detect human T lymphoblastic virus type 1 (HTLV1), and the real-time polymerase chain reaction is carried out in a single reaction mixture. The method of claim 7, wherein the probe combination is a probe having a fluorescent dye at the 5' end and a fluorescent inhibitory molecule at the 3' end.