WO2021051617A1 - 特异性检测样本中htlv-i前病毒dna的引物及其应用 - Google Patents

特异性检测样本中htlv-i前病毒dna的引物及其应用 Download PDF

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WO2021051617A1
WO2021051617A1 PCT/CN2019/118518 CN2019118518W WO2021051617A1 WO 2021051617 A1 WO2021051617 A1 WO 2021051617A1 CN 2019118518 W CN2019118518 W CN 2019118518W WO 2021051617 A1 WO2021051617 A1 WO 2021051617A1
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htlv
dna
sample
primer
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高歌
周安宇
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上海爱萨尔生物科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Definitions

  • the invention belongs to the field of molecular biological detection, and more specifically relates to the detection of HTLV-I proviral DNA in a sample.
  • HTLV-I Human T-lymphophilic virus type I
  • Oncovirus subfamily Mammal type C virus which can be divided into 6 subtypes, namely A to F. Under the electron microscope, they are spherical particles with a diameter of approximately It is 80 ⁇ 130nm and was discovered by American and Japanese scientists in the early 1980s. It is the first human retrovirus discovered [1].
  • the viral RNA can synthesize corresponding DNA through reverse transcription and integrate into the host genomic DNA to form a provirus (HTLV-I provirus) [2].
  • the cells infected by the virus are mainly CD4 + and CD8 + lymphocytes [3], but under certain conditions it can infect non-T lymphocytes, such as B lymphocytes, and even bone marrow stem cells and macrophages [4].
  • Human T-lymphophilic virus type I can cause a variety of diseases, such as adult T-cell leukemia (ATL) [5,6], tropical spastic lower limb paralysis (TSP) [7] and multiple sclerosis (MS), unexplained Vasculitis (KW) and so on.
  • ATL adult T-cell leukemia
  • TSP tropical spastic lower limb paralysis
  • MS multiple sclerosis
  • KW unexplained Vasculitis
  • HTLV-I is more carcinogenic than other carcinogenic viruses or bacteria (such as Epstein-Barr virus and Helicobacter pylori) [8].
  • diseases such as different blood-borne tumors, chronic pneumonia, rheumatoid arthritis, and certain solid tumors, which are probably caused by the human immune suppression caused by the infection of the HTLV-I virus.
  • HTLV-I human T-lymphophilic virus type I infection can induce immortalization of T cells; human T-lymphophilic virus type I infected umbilical cord T cells can continue to grow and express DR protein, and interleukin-2 receptor expression The amount increases, and the dependence on exogenous interleukin-2 decreases. These characteristics are similar to the characteristics of malignant T cells in ATL patients.
  • HTLV-I has two common methods of virus transmission. 1. Blood contact transmission: HTLV-I can be transmitted to the new host through infected T lymphocytes and macrophages during blood transfusion [9]. Second, breastfeeding transmission, HTLV-I can be transmitted to infants through breast milk [10], and there is also evidence that changing breastfeeding to formula feeding can effectively reduce the infection rate of infants with HTLV-I [11]. At present, there is no effective HTLV-I treatment and antiviral drugs, so the necessary prevention can only be carried out through HTLV-I testing.
  • HTLV-I testing methods include peripheral blood or bone marrow cytology, serum HTLV-I antibody testing, virus particle and antigen testing, and cerebrospinal fluid testing, such as ELISA (enzyme-linked immunosorbent assay), PA (particle agglutination), Serological diagnosis such as IF (immunofluorescence) and WB (immunoblotting), but these methods have certain limitations, such as the inability to distinguish between HTLV-I and HTLV-II infections, cumbersome operations, and low sensitivity and specificity. Prone to false positives.
  • Dye methods such as SYBR Green I dye can bind to the minor grooves of double-stranded DNA, and can produce fluorescent signals when excited, but since any double-strand can bind to it non-specifically to produce non-specific signals, it will cause inaccurate results.
  • the probe of the TaqMan probe method has a 5'-end fluorescent reporter group and a 3'-end quenching group. The complete probe undergoes fluorescence resonance energy transfer after being excited by the excitation light, so no signal can be detected; only when the DNA is replicated , The probe is hydrolyzed, and the reporter group and the quencher group are separated before the fluorescence can be detected.
  • the intensity of the fluorescence signal represents the number of templates, because the number of released fluorescent groups and the number of PCR products are a pair Therefore, this technique can be used to accurately quantify the template.
  • the SYBR Green and TaqMan technologies do not differ greatly in amplification efficiency [12]
  • the TaqMan technology is more suitable for the detection and quantification of micro-templates because of its higher specificity and higher sensitivity [13].
  • CN103898239B discloses a method for detecting HTLV-I and HTLV-II proviruses in the same tube with specific primers and probes.
  • a detection method with higher detection sensitivity to provide reliable preclinical diagnosis of HTLV-I evidence of.
  • the purpose of the present invention is to design and synthesize HTLV-I proviral DNA specific primers/probes, and develop a TaqMan qPCR method that can be used to detect HTLV-I proviral DNA in organs, tissues, cells, body fluids and blood.
  • This method It is suitable for detecting the presence of HTLV-I proviral DNA in the sample.
  • This method can qualitatively or quantitatively detect proviral DNA in infected HTLV-I cells in samples, with strong specificity, high precision and accuracy, and can stably detect very low levels of HTLV-I pre-virus in various samples. Viral DNA.
  • HTLV-I refers to human T lymphotropic virus type I, also known as human T lymphocytic leukemia virus type I, which is a retro RNA virus.
  • the RNA of the virus will synthesize the corresponding DNA through reverse transcription and integrate into the host genomic DNA, which is related to a variety of diseases.
  • the HTLV-I virus can be transmitted through blood.
  • HTLV-I proviral DNA refers to the DNA synthesized by reverse transcription and integrated into the host genome after the HTLV-I virus infects host cells.
  • the primer pair includes a forward primer 5'-CCCGACCTACCTATGGATAATGCT-3' (SEQ ID NO:1) and a reverse primer 5'-GGTTTTGTGGCAGTTGGTTAATACA -3' (SEQ ID NO: 2).
  • the primer pair includes a forward primer and a reverse primer, wherein the forward primer is 5'-CCCGACCTACCTATGGATAATGCT-3' (SEQ ID NO: 1), the reverse primer is 5'-GGTTTTGTGGCAGTTGGTTAATACA-3' (SEQ ID NO: 2); the sequence of the probe is 5'-CCCTATGGACAATCAACC-3' (SEQ ID NO: 3) ,
  • the 5'end of the probe is labeled with a reporter fluorophore, and the 3'end is labeled with a quencher group.
  • the reporter fluorophore labeled at the 5'end of the fluorescent probe is FAM
  • the quenching group labeled at the 3'end is NFQ-MGB or TAMRA.
  • Another aspect of the present invention provides the application of the aforementioned primer pair or primer and probe combination in the detection of HTLV-I proviral DNA in a sample.
  • Another aspect of the present invention provides the use of the aforementioned primer pair or primer and probe combination in preparing reagents for detecting HTLV-I proviral DNA in a sample.
  • the detection is by qPCR detection or by digital PCR detection.
  • qPCR also known as Real-time Quantitative PCR
  • the Ct value represents the cycle threshold, that is, the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold. Since the Ct value of each template has a linear relationship with the logarithm of the initial content of the template, the more the initial copy number, the smaller the Ct value.
  • a standard curve can be made using serially diluted standards with known initial content, where the abscissa represents the logarithm of the initial content, the ordinate represents the Ct value, or the ordinate represents the logarithm of the initial content, and the abscissa represents the Ct value. .
  • the content of the sample can be calculated from the standard curve.
  • qPCR is a mature technology in the field. When using existing instruments for qPCR detection, the Ct value of the sample can be directly obtained from the output result of the instrument.
  • fluorescent probes or fluorescent dyes can be used to obtain fluorescent signals.
  • a common fluorescent probe can be, for example, a TaqMan fluorescent probe, in which a specific fluorescent probe is added when a pair of primers are added during PCR amplification.
  • the probe is an oligonucleotide, and both ends are labeled with a reporter. Fluorophore and a quencher fluorophore.
  • the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme cleaves and degrades the probe, making the reporter fluorescent group and quencher
  • the fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, every time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product.
  • the reporter fluorophore group can be, for example, FAM
  • the quencher group can be, for example, NFQ-MGB.
  • fluorescent dyes can also be used to obtain fluorescent signals.
  • an excessive amount of fluorescent dyes can be added to the PCR reaction system. After the fluorescent dyes are non-specifically incorporated into the DNA double strands, they emit fluorescent signals without being incorporated into the strands. The dye molecule does not emit any fluorescent signal, thereby ensuring that the increase of the fluorescent signal is completely synchronized with the increase of the PCR product.
  • fluorescent dyes can be, for example, SYBR fluorescent dyes, sulforhodamine (Texas Red), fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), JOE, VIC, ROX And NED etc.
  • Digital PCR detection is also well known to those skilled in the art.
  • digital PCR also called single-molecule PCR
  • the sample is diluted to the single-molecule level and distributed evenly.
  • the reaction is carried out in tens to tens of thousands of units, and the fluorescent signal of each reaction unit is collected after the amplification is completed.
  • the original concentration or content of the sample is calculated by direct counting or Poisson distribution formula.
  • Those skilled in the art know how to perform digital PCR detection.
  • kits for specifically detecting HTLV-I proviral DNA in a sample includes the above-mentioned primer pair or a combination of the above-mentioned primer and probe.
  • the kit also includes any one or more reagents required for qPCR detection.
  • any one or more reagents required for qPCR detection include one or more components selected from the following components: qPCR reaction solution (for example, qPCR Master Mix (2X) , Including essential components such as enzymes required for qPCR reaction), nuclease-free high-purity water, positive control substance, negative control substance.
  • qPCR reaction solution for example, qPCR Master Mix (2X) , Including essential components such as enzymes required for qPCR reaction
  • nuclease-free high-purity water for example, qPCR Master Mix (2X) , Including essential components such as enzymes required for qPCR reaction
  • positive control substance can be genomic DNA or cells with HTLV-I proviral DNA.
  • the negative control substance can be genomic DNA or cells without HTLV-I proviral DNA.
  • the cell with HTLV-I proviral DNA may be, for example, an MT4 cell with HTLV-I proviral DNA, and the cell without HTLV-I proviral DNA may be, for example, a MOLT4 cell without HTLV-I proviral DNA.
  • Another aspect of the present invention provides a method for detecting HTLV-I proviral DNA in a sample, which includes using the aforementioned primer and probe combination to perform qPCR detection on the genomic DNA of the sample, and qualitatively or quantitatively detect the presence of the sample according to the qPCR detection result HTLV-I proviral DNA.
  • Another aspect of the present invention provides a method for detecting HTLV-I proviral DNA in a sample, which includes the following steps:
  • the Ct value of the DNA standard is used as the ordinate (Y), and the concentration of the genomic DNA with HTLV-I proviral DNA in the DNA standard is compared to The number is the abscissa (X), which fits the linear equation.
  • the aforementioned DNA standard contains at least 6 samples of different given concentrations prepared with genomic DNA with HTLV-I proviral DNA.
  • the number of DNA standards can be 6, 7, 8, or more.
  • the DNA standard can be prepared by mixing the genomic DNA of MT4 of HTLV-I positive cells and the genomic DNA of MOLT4 of HTLV-I negative cells at different concentrations, for example.
  • the highest concentration point refers to the highest limit of the concentration that can be stably detected by the above method, and can also be called the upper limit of detection or the upper limit of quantification in the present invention.
  • the lowest limit of the concentration that can be stably detected is the lowest point of concentration, which can also be referred to as the lower limit of detection or the lower limit of quantification in the present invention.
  • the qPCR reaction system is PCR Master Mix (2 ⁇ ) 10 ⁇ L, primers/probes (20 ⁇ ) specifically amplifying HTLV-I proviral DNA 1 ⁇ L, DNA sample + nuclease-free high-purity water 9 ⁇ L.
  • the DNA sample can be sample genomic DNA, DNA standard, or other positive control, negative control, or quality control.
  • the procedure of the qPCR reaction is to first activate UDG at 50°C for 2 minutes; then activate DNA polymerase at 95°C for 10 minutes; then perform 40 PCR reactions according to the following parameters: 95°C for 15 seconds; 60°C for 1 minute .
  • the qPCR reaction is completed on the Applied Biosystems ABI 7500 Real Time PCR machine.
  • the result determination may also be the quantitative detection of HTLV-I proviral DNA in the sample, or may further include the quantitative detection of HTLV-I proviral DNA in the sample.
  • the quantitative detection may include, for example, determining the concentration of genomic DNA with HTLV-I proviral DNA in the genomic DNA of the sample based on the Ct value of the genomic DNA of the sample and a fitted standard curve.
  • the sample may be an organ, tissue, whole blood, cell or body fluid sample.
  • the sample can be derived from humans or any animal, such as mice, rats, rabbits, monkeys and other animals.
  • the methods of the invention are performed in vitro.
  • the methods of the invention are non-diagnostic.
  • HTLV-I positive containing HTLV-I proviral DNA
  • HTLV-I infected can be used interchangeably, referring to those infected with HTLV-I, and the genomic DNA contains HTLV- I proviral DNA cells or tissues and organs containing these cells.
  • the HTLV-I specific primers and labeled probes provided by the present invention can be used for quantitative analysis of HTLV-I proviral DNA in tissues and blood, and can stably detect 1/10000 infection by HTLV-I virus within 1.5 hours
  • the cells of HTLV-I are of great significance for the preclinical diagnosis of HTLV-I and to determine whether it is infected with the HTLV-I virus, and will play an important role in the clinical test of HTLV-I virus.
  • the method of the invention not only has a simple preparation process and less time-consuming, but also has high detection accuracy, strong specificity and good repeatability.
  • Figure 1 is a simple experimental procedure of TaqMan-qPCR.
  • Figure 2 is a graph showing the amplification curve of qPCR detection with HTLV-I specific primers/probes.
  • Figure 3 is a standard curve diagram of qPCR detection with HTLV-I specific primers/probes. Wherein R 2 ⁇ 0.99.
  • the standard curve in the figure is fitted with Ct as the ordinate (Y) and the logarithm of the concentration of the standard concentration sample as the abscissa (X), but the numbers in the abscissa are still presented as the concentration of the standard concentration sample.
  • Figure 4 is an amplification curve diagram of a precision and accuracy verification experiment for qPCR detection using HTLV-I specific primers/probes.
  • Figure 5 is a graph showing the specific amplification curve of qPCR detection with HTLV-I specific primers/probes.
  • Figure 6 is a graph showing the dilution linear amplification curve of qPCR detection with HTLV-I specific primers/probes.
  • primers and labeled probes are designed and synthesized according to the STS sequence in the pol gene of human HTLV-I, and a standard curve is prepared with MT4 genomic DNA of HTLV-I positive cells to establish whether various samples are infected with HTLV-I
  • This method is used to perform comprehensive methodological verification of standard curve and quantitative range, accuracy and precision, dilution linearity, specificity, etc.
  • the experimental process is shown in Figure 1.
  • the STS sequence of the pol gene of human HTLV-I was used as a template to design and synthesize primers and fluorescently labeled probes.
  • the primer information is shown in Table 1:
  • the 5'end of the probe is labeled with the reporter fluorescent group FAM, and the 3'end is labeled with the quencher group NFQ-MGB.
  • the genomic DNA of the sample was extracted according to the standard procedures provided by the kit.
  • the qPCR reaction system was 20 ⁇ L, as shown in Table 2 below.
  • genomic DNA as a template, add Universal PCR Master Mix (2 ⁇ ), human HTLV-I specific primers and fluorescently labeled probes were used to complete the qPCR reaction on the Applied Biosystems ABI 7500 Real Time PCR machine.
  • the qPCR reaction conditions are shown in Table 3 below.
  • the MT4 genomic DNA of HTLV-I positive cells was serially diluted, and the HTLV-I specific primers and probes described in Example 1 were used for qPCR detection until the lowest limit of stable detection was 20pg/5 ⁇ l, which was set as the lower limit of detection (Or called the lower limit of quantification); the highest detectable limit is 90000pg/5 ⁇ l, which is set as the upper limit of detection (or called the upper limit of quantification); concentrations higher than the upper limit of detection can also be detected, but it may affect the accuracy of quantitative detection May increase the possibility of false positive results.
  • HTLV-I positive cell MT4 (purchased from Cybertron Biotechnology Co., Ltd.) genomic DNA mixed with HTLV-I negative cell MOLT4 (purchased from Cybertron Biotechnology Co., Ltd.) genomic DNA to prepare a standard concentration sample, including For 8 standard concentration samples, the DNA concentration is shown in Table 4 below. Dilute the negative cell MOLT4 genomic DNA to 20ng/ ⁇ L with DNase/RNase-Free water. The diluted negative cell MOLT4 genomic DNA is used to determine the MT4 genomic DNA concentration. Scalar dilution. The standard concentration sample preparation table is shown in Figure 4 below. The HTLV-I specific primers and probes described in Example 1 were used to perform qPCR detection on these standard concentration samples, and each sample was set with two multiple wells. The amplification curve is shown in FIG. 2.
  • the HTLV-I copy number is calculated as follows: the DNA quality of each MT4 cell is about 6.4 pg, and the HTLV copy number is 7, and the HTLV-I copy number is calculated based on the quality of the sample DNA.
  • the copy numbers of HTLV-I below are calculated in the same way.
  • the Ct value of the standard concentration sample is taken as the ordinate (Y), and the logarithm of the concentration of the standard concentration sample is taken as the abscissa (X), and the standard curve is fitted, as shown in Figure 3; the standard curve regression equation is obtained.
  • the correlation coefficient is shown in the table below.
  • the genomic DNA of MT4 cells containing HTLV-I proviral DNA and MOLT4 cells without HTLV-I proviral DNA were extracted. Dilute MOLT4 genomic DNA to 20ng/ ⁇ L with water as a negative control, and dilute MT4 cell genomic DNA to different concentrations to make strong positive control and critical positive control samples, plate quality control samples and precision accuracy samples, including strong positive control and critical positive
  • the control sample is used to evaluate whether the precision and accuracy of the qPCR experiment within a batch are acceptable.
  • the plate quality control samples are used to evaluate whether the stability of the qPCR experiment between different test batches is acceptable.
  • Table 5-7 is the preparation table of control samples, plate quality control samples and precision accuracy samples:
  • P-HQC, P-MQC, and P-LQC are quality control samples with high, medium, and low concentrations, respectively.
  • LLOQ is the Lower limit of quantification
  • LQC is Low Quality control, which is the same as P-LQC in Table 6
  • HQC High Quality control, as shown in Table 6.
  • the medium P-HQC is the same
  • MQC is the middle quality control (Middle Quality control), which is the same as the P-MQC in Table 6
  • ULOQ is the upper limit of quantification (ULOQ, Upper limit of quantification).
  • Genomic DNA was extracted from MT4 cells containing HTLV-I proviral DNA, and the lower limit of quantification (LLOQ, Lower limit of quantification), low concentration quality control (LQC, Low Quality control), and high concentration quality control (HQC, High Quality) were prepared separately Control), middle quality control (MQC, MiddleQuality control), and upper limit of quantification (ULOQ, Upper limit of quantification) samples with 5 concentrations.
  • the specific concentrations are shown in Table 7.
  • the HTLV-I specific primers and probes as described in Example 1 were used for qPCR detection to investigate the precision and accuracy of samples with different concentrations within and between batches.
  • the amplification curve is shown in FIG. 4. Accept the guidelines for verification of quantitative analysis methods of biological samples in the standard reference pharmacopoeia [15-17], as shown in Table 8
  • RE% (C measured- C theory or C 0 )/C theory or C 0 ⁇ 100%; where C 0 is the initial concentration or the theoretical concentration, which can be a known concentration or Concentration determined by other methods (the same below).
  • CV% standard deviation/average value ⁇ 100% (the same below).
  • the results are shown in Table 9:
  • the HTLV-I primer of Example 1 can stably detect 22 copies of the HTLV-I gene (approximately 20 pg of genomic DNA), and the intra-assay CV of the precision accuracy sample is -16.24% ⁇ 68.16%, inter-assay CV ranged from-11.56% to 53.18%, both in the range of -75% to 150%; intra-assay RE was 2.62% to 57.91%, inter-assay RE was 7.69% to 40.51%, which met the acceptance criteria and proved
  • the method for qPCR detection of HTLV-I provirus by the HTLV-I specific primer has high precision and accuracy, and the method has high sensitivity.
  • MOLT4 cells that do not carry HTLV-I provirus DNA 1Human MT4 cells carrying HTLV-I provirus DNA, 2MOLT4 cells that do not carry HTLV-I provirus DNA, 3No RNase/DNase Water, respectively extract the total genomic DNA, use the primers and probes described in Example 1 to perform qPCR detection, each group is equipped with two multiple wells, the amplification curve is shown in Figure 5.
  • the acceptance criteria are shown in Table 10 below:
  • the group added with human MT4 cells can detect the signal within the quantitative range, and the measured values of the other two groups are all lower than the lower limit of detection. This indicates that the HTLV-I primer can specifically recognize the DNA of cells infected with HTLV-I, and this method can accurately detect whether the sample contains cells infected with HTLV-I.
  • the genomic DNA of MT4 cells carrying HTLV-I proviral DNA was diluted to 3 ⁇ g/ ⁇ L with RNase/DNase-free water. After that, they were diluted 2 times, 10 times, 100 times and 1000 times respectively with RNase/DNase-free water, and the HTLV-I specific primers and probes described in Example 1 were used for TaqMan qPCR detection. Set two replicate wells in the group, repeat 3 times for each concentration, and the amplification curve is shown in Figure 6. The acceptance criteria are shown in Table 12 below:
  • HTLV-I specific primers and probes can sensitively detect 1/10000 infected cells in the cell population.
  • MT4 cells with HTLV-I proviral DNA positive control substance
  • MOLT4 cells without HTLV-I proviral DNA negative control
  • Example 1 Use the method of Example 1 to extract genomic DNA from the sample in step 1.
  • step 2 Take the genomic DNA obtained in step 2 as a template. Adjust the DNA content of the negative control substance to 50ng/5 ⁇ L, and adjust the initial DNA content of the positive control substance to 50ng/5 ⁇ L, and dilute to 1ng/5 ⁇ L, 0.2ng/5 ⁇ L, 0.04ng/5 ⁇ L, 0.008ng/5 ⁇ L, etc. The content serves as a control. The content of 3 umbilical cord DNA samples was adjusted to 50ng/5 ⁇ L for detection, and the result was judged according to the standard curve made in Example 2.
  • the qPCR reaction system is 20 ⁇ L, including: Universal PCR Master Mix (2X) 10 ⁇ L, HTLV-I proviral DNA (20x) primer/probe 1 ⁇ L, DNA sample and water total 9 ⁇ L.
  • reaction conditions were: 50°C, 2min; 95°C, 10min; then 40 PCR reactions were performed according to the following parameters: 95°C, 15 seconds, 60°C, 1min.
  • control primers are the primers for detecting HTLV-I in the patent CN103898239B described in the background technology section, and the primers described in Example 1 were synthesized, and The primers for detecting HTLV-I in the patent CN103898239B were verified by simultaneous qPCR experiments, and the detection sensitivity of the two methods was compared and analyzed.
  • the primers for detecting HTLV-I in the synthetic patent CN103898239B the sequence is as follows:
  • the fluorescent group labeled at the 5'end of the probe is JOE, and the quenching group labeled at the 3'end is TAMRA;
  • the ADK-HTLV-I primer in patent CN103898239B is compared with the primer in Example 1.
  • the Ct value of the ADK-HTLV-I primer is 5 higher Around the cycle, that is, the sensitivity is 30-40 times lower.
  • the primer in patent CN103898239B cannot detect HTLV-I in the sample, and the primer in Example 1 is used in the starting DNA.
  • the HTLV-I proviral DNA in the sample can still be detected.
  • the experimental process is simple, convenient, accurate, sensitive and specific.
  • IFA indirect immunofluorescence
  • GPA gelatin particle agglutination
  • ELISA enzyme-linked immunosorbent assay
  • WB Western blotting
  • Gallo R C The discovery of the first human retrovirus: HTLV-1 and HTLV-2[J].Retrovirology,2005,2(1):17.
  • HTLV-I Human T-cell leukemia virus type I
  • ATL adult T-cell leukemia
  • Nguyen PK Riegler J, Wu JC. Stem cell imaging: from bench to bedside. Cell Stem Cell. 2014; 14(4): 431-444.

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Abstract

提供特异性检测HTLV-I前病毒DNA的引物和荧光探针，以及检测样本中HTLV-I前病毒DNA的TaqMan qPCR方法，可用于定性或定量检测样本中的感染HTLV-I细胞中的前病毒DNA。

Description

特异性检测样本中HTLV-I前病毒DNA的引物及其应用 技术领域
本发明属于分子生物检测领域，更具体涉及对样本中HTLV-I前病毒DNA的检测。
背景技术
人嗜T淋巴病毒I型(HTLV-I)属于逆转录病毒科肿瘤病毒亚科哺乳类C型病毒，又可以分为6种亚型，分别为A～F，电镜下呈球形颗粒，直径约为80～130nm，在20世纪80年代初被美国和日本科学家发现，是最早发现的一种人类逆转录病毒[1]。
人嗜T-淋巴病毒I型感染宿主细胞后，病毒的RNA能够经逆转录合成相应DNA并整合进入宿主基因组DNA形成前病毒(HTLV-I provirus)[2]。该病毒感染的细胞主要是CD4 +和CD8 +淋巴细胞[3]，但在特定条件下可感染非T淋巴细胞，如B淋巴细胞，甚至可感染骨髓干细胞和巨噬细胞[4]。
人嗜T-淋巴病毒I型能引起多种疾病，如成人T细胞白血病(ATL)[5,6]、热带痉挛性下肢瘫(TSP)[7]和多发硬化症(MS)、不明原因的脉管炎(KW)等。有研究认为，HTLV-I相较于其他致癌病毒或致癌细菌(如EB病毒和幽门螺旋杆菌)具有更强的致癌性[8]。还有一些疾病，如不同的血源性肿瘤、慢性肺炎疾患、风湿性关节炎和某些实质性肿瘤等，很可能也是由于感染HTLV-I病毒后产生的人体免疫抑制所导致的疾病。目前，全球有大于2000万人感染HTLV-I，该病毒伴有较长的潜伏期，发病较为缓慢，约有2-5％的感染者一直会表现出潜伏无症状期。但约0.1-2％的HTLV-I感染者比例将发展为上述临床症状[6]。在细胞培养上，人嗜T-淋巴病毒I型感染可诱导T细胞永生化；人嗜T-淋巴病毒I型感染的脐带T细胞可持续生长并表达DR蛋白，白细胞介素-2受体表达量增加，而对外源性白细胞介素-2的依赖性降低，这些特性与ATL患者的恶性T细胞特性相似。
HTLV-I有两种常见的方法病毒传播方式。一、血液接触传播：输血时HTLV-I可通过被感染T淋巴细胞和巨噬细胞传播到新宿主中[9]。二，母乳喂养传播，HTLV-I能通过母乳传播至婴儿[10]，也有证据表明，将母乳喂养改为配方奶喂养能够有效降低婴儿HTLV-I感染率[11]。目前，尚无有效的HTLV-I治疗方法及抗病毒药物，因此只能通过HTLV-I检测进行必要预防。现有的HTLV-I的检测方法包括外周血或骨髓细胞学检查、血清HTLV-I抗体检测、病毒颗粒及抗原检测和脑脊液检查，例如ELISA(酶联免疫吸附检测)、PA(颗粒凝集)、IF(免疫荧光)、WB(免疫印迹)等血清学诊断，但这些方法有一定的局限性，如不能区分是HTLV-Ⅰ还是HTLV-Ⅱ的感染，操作繁琐，灵敏性和特异性不高，容易出现假阳性。
实时荧光定量PCR(qRT-PCR，quantitative real time polymerase chain reaction)技术的发展实现了PCR从定性到定量的飞跃，而且其特异性强、灵敏度高、检测方法简便快速、能有效检测出低拷贝的目的DNA片段，为我们提供了新的检测HTLV-I手段。应用实时荧光定量PCR技术并设计针对整合于宿主基因组中的HTLV-I前病毒DNA片段的特异引物、探针，理论上能够大大提高检测样本中HTLV-I的灵敏性和特异性，避免目前已有的HTLV-I检测手段的不足之处。
qRT-PCR技术有两种方法：染料法和TaqMan探针法。染料法如SYBR Green I染料可与双链DNA的小沟结合，当被激发后可以产生荧光信号，但由于任何双链都可以与其非特异性结合产生非特异性信号，因此会造成不准确的结果。TaqMan探针法的探针具有5’端荧光报告基团和3’端淬灭基团，完整的探针受到激发光后会发生荧光共振能量转移，因此检测不到信号；只有当DNA复制时，探针被水解，报告基团和淬灭基团分离，才可以检测到荧光，因此荧光信号的强弱就代表了模板的数量，由于被释放的荧光基团数目和PCR产物数量是一对一的关系，因此用该技术可对模板进行准确定量。虽然SYBR Green和TaqMan技术在扩增效率上差别不是很大[12]，但TaqMan技术因为特异性更高、敏感性更强，更适用于微量模板的检测和定量[13]。
CN103898239B公开了一种用特异性引物和探针同管检测HTLV-I和HTLV-II前病毒的方法，但是，仍然需要开发检测灵敏度更高的检测方法，以为HTLV-I的临床前诊断提供可靠的证据。
发明内容
本发明的目的是设计并合成HTLV-I前病毒DNA特异性的引物/探针，及开发可用于检测器官、组织、细胞、体液和血液中HTLV-I前病毒DNA的TaqMan qPCR方法，此方法适用于检测样本中是否存在HTLV-I前病毒DNA。该方法可以定性或定量检测样本中的感染HTLV-I细胞中的前病毒DNA，特异性强、精密度和准确度高，并可以稳定检测到各类样本中的极低含量的HTLV-I前病毒DNA。
术语“HTLV-I”是人嗜T淋巴病毒I型，也被称为人类T淋巴细胞白血病病毒Ⅰ型，是一种逆转录RNA病毒。该病毒的RNA经逆转录会合成相应的DNA并整合进入宿主基因组DNA，与多种疾病相关。HTLV-I病毒可经由血液传播。
术语“HTLV-I前病毒DNA”是指HTLV-I病毒感染宿主细胞后，逆转录合成并整合到宿主基因组上的DNA。
本发明一方面提供特异性检测样本中HTLV-I前病毒DNA的引物对，所述引物对包括正向引物5'-CCCGACCTACCTATGGATAATGCT-3'(SEQ ID NO:1)和反向引物5'-GGTTTTGTGGCAGTTGGTTAATACA-3'(SEQ ID NO:2)。
本发明的另一方面提供特异性检测样本中HTLV-I前病毒DNA的引物和探针组合，所述引物对包括正向引物和反向引物，其中正向引物是5'-CCCGACCTACCTATGGATAATGCT-3'(SEQ ID NO:1)，所述反向引物是5'- GGTTTTGTGGCAGTTGGTTAATACA-3'(SEQ ID NO:2)；所述探针的序列为5'-CCCTATGGACAATCAACC-3'(SEQ ID NO:3)，该探针的5’端标记报告荧光基团，3’端标记淬灭基团。
在一些实施方案中，所述荧光探针的5’端标记的报告荧光基团为FAM，3’端标记的淬灭基团为NFQ-MGB或TAMRA。
本发明的另一方面提供上述引物对或引物和探针组合在检测样本中HTLV-I前病毒DNA中的应用。
本发明的另一方面提供上述引物对或引物和探针组合在制备检测样本中HTLV-I前病毒DNA的试剂中的应用。
在一些实施方案中，所述检测是通过qPCR检测或通过数字PCR检测。
如本领域技术人员所知，qPCR又称实时定量PCR(Real-time Quantitative PCR)，是在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测PCR进程，最后可以通过标准曲线对未知模板进行定量分析。在qPCR检测中，Ct值表示循环阈值，即每个反应管内的荧光信号达到设定阈值时所经历的循环数。由于每个模板的Ct值与该模板的起始含量的对数存在线性关系，起始拷贝数越多，Ct值越小。利用连续稀释的已知起始含量的标准品可作出标准曲线，其中横坐标代表起始含量的对数，纵坐标代表Ct值，或者纵坐标代表起始含量的对数，横坐标代Ct值。只要获得未知样品的Ct值，即可从标准曲线上计算出该样品的含量。qPCR在本领域中属于成熟技术，利用现有的仪器进行qPCR检测时，可以直接从仪器的输出结果中获得样本的Ct值。
在qPCR检测中，可以使用荧光探针或荧光染料获取荧光信号。常见的荧光探针例如可以是TaqMan荧光探针，其中PCR扩增时在加入一对引物的同时加入一个特异性的荧光探针，该探针为一寡核苷酸，两端分别标记一个报告荧光基团和一个淬灭荧光基团。探针完整时，报告基团发射的荧光信号被淬灭基团吸收；PCR扩增时，Taq酶的5'－3'外切酶活性将探针酶切降解，使报告荧光基团和淬灭荧光基团分离，从而荧光监测系统可接收到荧光信号，即每扩增一条DNA链，就有一个荧光分子形成，实现了荧光信号的累积与PCR产物形成完全同步。在一些实施方案中，报告荧光基团可以是例如FAM，淬灭基团可以是例如NFQ-MGB。本领域技术人员知道其它报告荧光基因和相应的淬灭荧光基团也可以用于本发明。
在qPCR检测中，还可以使用荧光染料获取荧光信号，例如可以在PCR反应体系中，加入过量荧光染料，荧光染料非特异性地掺入DNA双链后，发射荧光信号，而不掺入链中的染料分子不会发射任何荧光信号，从而保证荧光信号的增加与PCR产物的增加完全同步。常用的荧光染料例如可以是SYBR荧光染料、磺酰罗丹明(Texas Red)、异硫氰酸荧光素(FITC)、羟基荧光素(FAM)、四氯荧光素(TET)、JOE、VIC、ROX和NED等。
数字PCR检测也是本领域技术人员公知的，简言之，数字PCR(也可称单分子PCR)包括PCR扩增和荧光信号分析，在PCR扩增阶段，将样品稀释到单分子水平并 平均分配到几十至几万个单元中进行反应，在扩增结束后对每个反应单元的荧光信号进行采集。最后通过直接计数或泊松分布公式计算得到样品的原始浓度或含量。本领域技术人员熟知如何进行数字PCR检测。
本发明的另一方面提供特异性检测样本中HTLV-I前病毒DNA的试剂盒，该试剂盒包括上述引物对，或包括上述引物和探针组合。
在一些实施方案中，所述试剂盒中还包括进行qPCR检测所需要的任意一种或更多种试剂。
在一些实施方案中，所述进行qPCR检测所需要的任意一种或更多种试剂包括选自下述组分的一种或更多中组分：qPCR反应液(例如qPCR Master Mix(2X)，其中包括qPCR反应所需要的酶等必须成分)、无核酸酶高纯水、阳性对照品、阴性对照品。其中阳性对照品可以是具有HTLV-I前病毒DNA的基因组DNA或细胞。其中阴性对照品可以是无HTLV-I前病毒DNA的基因组DNA或细胞。其中具有HTLV-I前病毒DNA的细胞例如可以是具有HTLV-I前病毒DNA的MT4细胞，其中无HTLV-I前病毒DNA的细胞例如可以是无HTLV-I前病毒DNA的MOLT4细胞。
本发明的另一方面提供样本中HTLV-I前病毒DNA的检测方法，包括使用上述的引物和探针组合，对样本基因组DNA进行qPCR检测，根据qPCR检测结果定性检测或定量检测样本中是否存在HTLV-I前病毒DNA。
本发明的另一方面提供样本中HTLV-I前病毒DNA的检测方法，包括以下步骤：
(1)提取样本基因组DNA；
(2)使用特异性扩增HTLV-I前病毒DNA的引物和探针组合，例如上述的引物和探针组合，对DNA标准品和样本基因组DNA进行qPCR检测，其中DNA标准品是用具有HTLV-I前病毒DNA的基因组DNA配制的不同给定浓度的样品；
(3)用DNA标准品的qPCR检测结果制作标准曲线，拟合线性方程，其中R 2≥0.99；且各标准品浓度的准确度(RE％)为-75％～150％，确定可稳定检测到的浓度最低点；
(4)结果判定：如果样本基因组DNA的qPCR结果中出现明显的扩增曲线；且样本基因组DNA的qPCR的Ct值小于浓度最低点的Ct值，则为阳性结果，即样本中存在HTLV-1前病毒DNA；如果无明显扩增曲线，或者有明显扩增曲线，但Ct值大于标准曲线浓度最低点的Ct值，则为阴性结果，即样本中不存在HTLV-I前病毒DNA。
在一些实施方案中，上述步骤(3)中制作标准曲线时，以DNA标准品的Ct值为纵坐标(Y)，以DNA标准品中具有HTLV-I前病毒DNA的基因组DNA的浓度的对数为横坐标(X)，拟合线性方程。
在一些实施方案中，上述DNA标准品包含至少6个用具有HTLV-I前病毒DNA的基因组DNA配制的不同给定浓度的样品。DNA标准品的数量可以是6个、7个、8个或更多个。DNA标准品例如可以用HTLV-I阳性细胞MT4的基因组DNA与HTLV-I阴性细胞MOLT4基因组DNA以不同浓度配比配制而成。
拟合标准曲线时，如R 2和各标准品浓度的准确度不满足以上要求，可以通过重复实验，或重新制备DNA标准品、或换用不同浓度的DNA标准品，来获得满意的拟合结果。标准曲线的拟合以及获得满意拟合结果的方法属于本领域技术人员的公知技术。
浓度最高点是指用上述方法可稳定检测到的浓度的最高限，在本发明中也可称为检测上限或定量上限。可稳定检测到的浓度的最低限即为浓度最低点，在本发明中也可称为检测下限或定量下限。
判断是否出现明显扩增曲线的方法是本领域技术人员公知的，例如当ΔRn vs Cycle模式下的曲线为S形时，可确定出现明显扩增曲线。
在一些实施方案中，qPCR的反应体系为PCR Master Mix(2×)10μL，特异性扩增HTLV-I前病毒DNA的引物/探针(20×)1μL，DNA样品+无核酸酶高纯水9μL。其中DNA样品可以是样本基因组DNA、DNA标准品、或其它阳性对照品、阴性对照品或质控品。
在一些实施方案中，qPCR反应的程序为首先50℃，2min以激活UDG；其次95℃，10min激活DNA聚合酶；然后按下列参数进行40个PCR反应：95℃，15秒；60℃，1min。在一些实施方案中，在Applied Biosystems ABI 7500Real Time PCR仪上完成qPCR反应。
在一些实施方案中，所述结果判定也可以是定量检测样品中的HTLV-I前病毒DNA，或者可以进一步包括定量检测样品中的HTLV-I前病毒DNA。所述定量检测可以包括例如根据样本基因组DNA的Ct值和拟合的标准曲线，确定样本基因组DNA中具有HTLV-I前病毒DNA的基因组DNA的浓度。
本发明中，样本可以是器官、组织、全血、细胞或体液样品。
本发明中，样本可以来源于人或任何动物，例如小鼠、大鼠、兔、猴等动物。
在一些实施方案中，本发明的方法是在体外进行的。
在一些实施方案中，本发明的方法是非诊断性的。
本申请中，术语“HTLV-I阳性的”、“含有HTLV-I前病毒DNA的”，“受HTLV-I感染的”可以交换使用，指受到HTLV-I感染的，基因组DNA中含有HTLV-I前病毒DNA的细胞或含有这些细胞的组织、器官。
本发明提供的HTLV-I特异性引物和标记探针可用于对组织、血液中的HTLV-I前病毒DNA进行定量分析，可以在1.5小时内稳定地检测到1/10000被HTLV-I病毒感染的细胞，为HTLV-I的临床前诊断、判断是否感染HTLV-I病毒具有重要意义，将在HTLV-I病毒临床检验中，发挥重要作用。本发明的方法不仅制备过程简单、耗时少，而且检测的准确度高、特异性强、重复性好。
附图说明
图1是TaqMan-qPCR简易实验流程。
图2是用HTLV-I特异性引物/探针进行qPCR检测的扩增曲线图。
图3是用HTLV-I特异性引物/探针进行qPCR检测的标准曲线图。其中R 2≥0.99。图中标准曲线以Ct值为纵坐标(Y)，以标准浓度样品的浓度的对数为横坐标(X)拟合而成，但横坐标中的数字仍以标准浓度样品的浓度呈现。
图4是用HTLV-I特异性引物/探针进行qPCR检测的精密度和准确度验证实验的扩增曲线图。
图5是用HTLV-I特异性引物/探针进行qPCR检测的特异性扩增曲线图。
图6是用HTLV-I特异性引物/探针进行qPCR检测的稀释线性扩增曲线图。
实施例
下面通过实施例，并结合附图，对本发明的技术方案作进一步详细的说明，但本发明不限于下面的实施例。
在以下实施例中，根据人HTLV-I的pol基因中的STS序列设计、合成引物和标记探针，以HTLV-I阳性细胞MT4基因组DNA配制标准曲线，建立鉴别各类样本是否感染HTLV-I的检测方法，并以该方法进行标准曲线与定量范围、准确度与精密度、稀释线性、特异性等的全面方法学验证，实验流程参见附图1。
如无特别说明，下述实施例中提及的序列均以从5’端到3’端的方式表示。
实施例1 引物设计与基因DNA提取方法
1.1引物的设计与合成
根据NCBI基因序列数据库提供的基因序列信息，以人HTLV-I的pol基因STS序列为模板设计并合成引物及荧光标记的探针，引物信息如下表1：
表1 引物设计相关信息表
引物名称 HTLV-I
Gene ID 1491936
STS GDB:456150
正向引物序列 CCCGACCTACCTATGGATAATGCT
反向引物序列 GGTTTTGTGGCAGTTGGTTAATACA
探针序列 CCCTATGGACAATCAACC
该探针的5’端标记报告荧光基团FAM，3’端标记淬灭基团NFQ-MGB。
1.2基因组DNA的提取
采用QIAGEN公司的基因组DNA提取试剂盒DNeasyBlood&Tissue Kit，按照试剂盒提供的标准程序提取样本基因组DNA。
将样本加入蛋白酶K裂解后，依次加入试剂盒内的各种缓冲液：buffer AL、无水乙醇、buffer AW1、buffer AW2等，通过DNeasy Mini spin离心柱离心富集基因组DNA，最后使用buffer AE溶解基因组DNA，用Nanodrop进行浓度和质量(A260/280)的测定后，保存于-80℃备用。
实施例2 荧光定量PCR方法及方法学验证
2.1荧光定量PCR反应条件
使用Taqman-qPCR方法，qPCR反应体系为20μL，如下述表2所示。以基因组DNA为模板，加入
Figure PCTCN2019118518-appb-000001
Universal PCR Master Mix(2×)、人HTLV-I的特异性引物和荧光标记的探针，在Applied Biosystems ABI 7500Real Time PCR仪上完成qPCR反应。qPCR反应条件见下述表3。
表2 qPCR板加样配比表
Figure PCTCN2019118518-appb-000002
表3 qPCR反应条件表
Figure PCTCN2019118518-appb-000003
如无特别说明，下述实施例均按照本2.1节中的反应体系、反应条件和说明进行qPCR检测。
2.2方法学验证
2.2.1制做标准曲线
对HTLV-I阳性细胞MT4基因组DNA进行梯度稀释，并用实施例1所述的HTLV-I特异性引物和探针进行qPCR检测，直至可稳定检测到的最低限为20pg/5μl，设为检测下限(或称定量下限)；可检测到的最高限，为90000pg/5μl，设为检测上限(或称定量上限)；高于检测上限的浓度也可以被检测到，但有可能影响定量检测的准确性或增加出现假阳性结果的可能性。
以HTLV-I阳性细胞MT4(购买于赛百慷生物技术股份有限公司)基因组DNA混合HTLV-I阴性细胞MOLT4(购买于赛百慷生物技术股份有限公司)基因组DNA配制成标准浓度样品，共包含8个标准浓度样品，其DNA浓度见下述表4，以DNase/RNase-Free water将阴性细胞MOLT4基因组DNA稀释至20ng/μL，稀释后的阴性细胞MOLT4基因组DNA用于对MT4基因组DNA浓度进行梯度稀释。标准浓度样 品配制表如下图4所示。用实施例1中所述的HTLV-I特异性引物和探针对这些标准浓度样品进行qPCR检测，每个样品设置两个复孔，扩增曲线图如图2所示。
表4 标准曲线样品配制表
Figure PCTCN2019118518-appb-000004
其中HTLV-I拷贝数按以下方式计算得到：每个MT4细胞的DNA质量大约6.4pg，携带HTLV拷贝数为7，根据样品DNA的质量计算出HTLV-I的拷贝数。下文中HTLV-I拷贝数均按同样的方式计算得到。
检测结束后以标准浓度样品的Ct值为纵坐标(Y)，以标准浓度样品的浓度的对数为横坐标(X)，拟合成标准曲线，如图3所示；得到标准曲线回归方程及相关系数如下表所示。
Figure PCTCN2019118518-appb-000005
2.2.2对照样本、板质控样本与精密度准确度样本的配制
提取含有HTLV-I前病毒DNA的MT4细胞和不含HTLV-I前病毒DNA的MOLT4细胞的基因组DNA。用水稀释MOLT4基因组DNA至20ng/μL后作为阴性对照，稀释MT4细胞基因组DNA至不同浓度做强阳性对照和临界阳性对照样本、板质控样本和精 密度准确度样本，其中强阳性对照和临界阳性对照样本用于评估一个批次内qPCR实验的精密度和准确度是否接受。板质控样本用于评估不同检测批次间qPCR实验的稳定性是否接受。以下表5-7为对照样本、板质控样本与精密度准确度样本配制表：
表5 对照样本配制表
Figure PCTCN2019118518-appb-000006
表6 板质控样本配制表
Figure PCTCN2019118518-appb-000007
表7 精密度准确度样本配制表
Figure PCTCN2019118518-appb-000008
表6中P-HQC、P-MQC、P-LQC分别为高、中、低三个浓度的质控样品。表7中LLOQ为定量下限(Lower limit of quantification)，LQC为低浓度质控(Low Quality control)，与表6中P-LQC相同，HQC为高浓度质控(High Quality control)，与表6中P-HQC相同，MQC为中浓度质控(MiddleQuality control)，与表6中P-MQC相同，ULOQ为定量上限(ULOQ，Upper limit of quantification)。
2.2.3精密度、准确度检测
从含有HTLV-I前病毒DNA的MT4细胞中提取基因组DNA，分别配制定量下限(LLOQ，Lower limit of quantification)、低浓度质控(LQC，Low Quality control)、 高浓度质控(HQC，High Quality control)、中浓度质控(MQC，MiddleQuality control)及定量上限(ULOQ，Upper limit of quantification)等5个浓度的样品，其具体浓度如表7所示。用如实施例1中所述的HTLV-I特异性引物和探针进行qPCR检测，考察不同浓度样品的批内及批间的精密度和准确度，其扩增曲线图如图4所示。接受标准参考药典中生物样品定量分析方法验证指导原则[15-17]，如表8所示：
表8 精密度、准确度检测接受标准
Figure PCTCN2019118518-appb-000009
其中准确度即RE％，计算公式：RE％＝(C 测得-C 理论or C 0)/C 理论or C 0×100％；其中C 0为初始浓度或理论浓度，可以是已知浓度或通过其它方法测定的浓度(下同)。
其中精密度即CV％，计算公式：CV％＝标准偏差/平均值×100％(下同)。
结果如表9所示：实施例1的HTLV-I引物可以稳定检测到22个拷贝的HTLV-I基因(约为20pg基因组DNA)，并且精密度准确度样品的批内CV为-16.24％～68.16％，批间CV为-11.56％～53.18％，均在-75％～150％之间；批内RE为2.62％～57.91％，批间RE为7.69％～40.51％，符合接受标准，证明通过该HTLV-I特异性引物对HTLV-I前病毒进行qPCR检测的方法具有较高的精密度与准确度，且该方法具备较高的灵敏性。
表9 准确度和精密度检测结果
Figure PCTCN2019118518-appb-000010
2.2.4特异性检测
在不携带HTLV-I前病毒DNA的MOLT4细胞中分别加入等体积的：①携带HTLV-I前病毒DNA的人MT4细胞，②不携带HTLV-I前病毒DNA的MOLT4细胞，③无 RNase/DNase水，分别提取基因组总DNA，用实施例1所述的引物和探针进行qPCR检测，每组设置两个复孔，扩增曲线如图5所示。接受标准如下表10所示：
表10 特异性检测接受标准
Figure PCTCN2019118518-appb-000011
结果如表11所示：加入人MT4细胞的组别可以在定量范围内检测到信号，其余两组测定值均低于检测下限。这表明该HTLV-I引物能够特异性地识别受HTLV-I感染的细胞DNA，利用该方法能够准确检测出样本中是否含有受HTLV-I感染的细胞。
表11 特异性检测结果
组别 CV％ 结果判定
加入MT4 4.4 高于定量下限
加入MOLT4 NA 低于定量下限
加入无RNase/DNase水 NA 低于定量下限
2.2.5稀释线性检测
为了进一步验证实施例1所述HTLV-I特异性引物及qPCR实验方法的精确性和有效性，使用无RNase/DNase水梯度稀释携带HTLV-I前病毒DNA的MT4细胞的基因组DNA至3μg/μL后，再以无RNase/DNase水对其分别进行2倍、10倍、100倍和1000倍的梯度稀释，用实施例1所述的HTLV-I特异性引物和探针进行TaqMan qPCR检测，每组设置两个复孔，每个浓度重复3次，扩增曲线如图6所示。接受标准如下表12所示：
表12 稀释线性检测接受标准
Figure PCTCN2019118518-appb-000012
结果如表13所示：所有浓度来源的稀释样品回算浓度后，其CV为4.2％～17.0％，均≤60％，证明使用该HTLV-I特异性引物进行qPCR检测能够精确检测到样本中的HTLV-I基因组DNA。
表13 稀释线性检测结果
组别 稀释倍数 CV％
DIL1 2 4.2
DIL2 10 7.0
DIL3 100 9.5
DIL4 1000 17.0
2.2.6细胞样本中不同比例的HTLV-I感染细胞的检测
取携带HTLV-I前病毒DNA的MT4细胞和不含HTLV-I前病毒DNA的MOLT4细胞。混合不同比例的MT4细胞和MOLT4细胞后提取基因组DNA，用实施例1所述的HTLV-I特异性引物和探针进行qPCR检测，混合比例与接受标准如下表14所示：
表14 细胞样本中不同比例的HTLV-I感染细胞的检测接受标准
Figure PCTCN2019118518-appb-000013
结果如表15所示：HTLV-I特异性引物及探针可以敏感性地检测到细胞群中1/10000被感染的细胞。
表15 细胞样本中的不同浓度的HTLV-I感染细胞选择性检测结果
组别 MT4细胞数(个) MOLT4细胞数(个) MT4细胞比例 检测结果
1 4000 396000 1/100 阳性
2 2000 398000 1/200 阳性
3 400 399600 1/1000 阳性
4 200 399800 1/2000 阳性
5 40 399960 1/10000 阳性
6 0 400000 0 阴性
实施例3 用TaqMan qPCR检测脐带组织中是否存在HTLV-I前病毒DNA
一、样品来源
3个脐带组织块样本；
具有HTLV-I前病毒DNA的MT4细胞：阳性对照品；
无HTLV-I前病毒DNA的MOLT4细胞：阴性对照品；
二、基因组DNA提取
使用实施例1的方法对步骤一中的样品提取基因组DNA。
三、TaqMan qPCR检测
以步骤二获得的基因组DNA为模板。将阴性对照品的DNA含量调节为50ng/5μL，阳性对照品的起始DNA含量调节为50ng/5μL，并稀释为1ng/5μL、0.2ng/5μL、0.04ng/5μL、0.008ng/5μL等不同含量做为对照。将3个脐带DNA样本含量调节为50ng/5μL进行检测，根据实施例2制作的标准曲线进行结果判断。
qPCR反应体系为20μL，包括：
Figure PCTCN2019118518-appb-000014
Universal PCR Master Mix(2X)10μL、HTLV-I前病毒DNA(20x)引物/探针1μL、DNA样本和水共9μL。
反应条件为：50℃，2min；95℃，10min；然后按下列参数进行40个PCR反应：95℃，15秒，60℃，1min。
结果如表16所示，其中显示在50到0.008ng的阳性对照中均检测到HTLV-I前病毒DNA的扩增，且检测到的DNA片段拷贝数与样品DNA含量成正相关，而在阴性对照DNA中和3个脐带样品中均未检测到HTLV-I前病毒DNA的扩增，提示检测的3个脐带组织样本均未感染HTLV-I病毒。
表16 qPCR检测脐带组织中HTLV-I前病毒DNA结果
样本编号 样本名称 Ct值 结果判断
1 MT4-50ng 23.575 阳性
2 MT4-1ng 29.972 阳性
3 MT4-0.2ng 32.713 阳性
4 MT4-0.04ng 36.014 阳性
5 MT4-0.008ng 36.838 阳性
6 MOLT4-50ng 未检测到 阴性
7 18号脐带 未检测到 阴性
8 19号脐带 未检测到 阴性
9 20号脐带 未检测到 阴性
实施例4 与现有的HTLV-I检测方法的对比
为了验证用本发明的引物进行qPCR方法的高效性，我们进行了对比实验，对照引物是背景技术部分所述的专利CN103898239B中检测HTLV-I的引物，合成上述实施例1所述的引物，以及专利CN103898239B中检测HTLV-I的引物，进行同步qPCR实验进行验证，对比并分析两种方法的检测灵敏度。
4.1引物合成
合成专利CN103898239B中检测HTLV-I的引物，序列如下：
引物名称 ADK-HTLV-I
正向引物序列 CCCATTGGCTCCTGTGAAAG
反向引物序列 TTGAGACAGCGCCACGAAC
探针序列 TCTCGGACTTTTCCATGGCCTCTCC
其中探针5’端标记荧光基团为JOE，3’端标记淬灭基团为TAMRA；
4.2实验方法
按照“实施例2荧光定量PCR方法及方法学验证”方法中的“2.2.1制做标准曲线”稀释MT4细胞DNA样品并配制标准曲线，并按照“2.1荧光定量PCR反应条件”配制混合管并设置qPCR反应的程序。
4.3实验结果
实验过程中除引物不同之外，其余全部按照上述“4.2实验方法”所述制作标准曲线，并使用相同的反应程序进行qPCR检测。
实验结果如下表所示：
Figure PCTCN2019118518-appb-000015
从上表数据可以看出：专利CN103898239B中的ADK-HTLV-I引物与实施例1的引物相对比，在初始DNA模板含量相同的条件下，ADK-HTLV-I引物的Ct值要高5个循环左右，即灵敏度要低30-40倍，当起始DNA低于或等于160pg时，使用专利CN103898239B中的引物不能检测到样本中的HTLV-I，而使用实施例1的引物在起始DNA为20pg时，仍能检测到样品中的HTLV-I前病毒DNA。
以上结果表明，相对于专利CN103898239B中的HTLV-I引物，本发明中所使用的引物/探针和TaqMan qPCR实验方法的灵敏度高、特异性强，更有利于检测HTLV-I的感染。
结论
以人HTLV-I特异性序列设计并合成的上述引物、标记探针，结合TaqMan qPCR实验技术，可以特异性检测不同类型样本是否存在HTLV-I感染。实验过程简单、方便，准确度高，灵敏性和特异性强。
讨论
HTLV-I的传播方式主要有两种，包括血液传播和母婴传播，此外还有性接触传播，共用针头传播等[14]。目前，尚无有效的治疗药物，因此只能通过HTLV-I筛查进行必要预防。从根本上说,治愈所有传染病的良方在预防感染方面。美国、西欧、日本、日本、香港等国家和地区均已在献血员和血液制品中实施HTLV-I筛查，以切断其传播途径。而中国对HTLV-I筛查落后了一步，感染HTLV-I是一种慢性破坏性疾病，因此对人员进行相关的HTLV-I筛查很有必要。
目前除了传统的细胞学检查外，对HTLV-I的检测多采用间接免疫荧光法(IFA)、明胶颗粒凝集反应(GPA)、酶联免疫吸附试验(ELISA)及蛋白印迹试验(WB)等。但是基于抗原、抗体的免疫检测具有操作繁琐、过程不易标准化、灵敏度低、假阳性高、成本高等缺点。国内相关检测技术及检测试剂稀少，因此需要新的检测方法的出现解决上诉问题。
在本实验中，我们设计并合成了人特异性序列HTLV-I的引物、探针，利用TaqMan qPCR技术，可以对样本中的HTLV-I感染细胞进行特异性检测。基于对引物和TaqMan qPCR技术进行特异性、稀释线性、精密度和准确度等的验证，证明本方法可以灵敏的检测各类样本的HTLV-I前病毒基因组DNA。不同于其它有不足或缺陷的检测手段，我们的方法具有很高的精确度和重复性，而且灵敏度高，可以稳定检测到细胞群中1/10000被感染的细胞。因此，我们的HTLV-I引物及TaqMan PCR技术可以应用于检测各类样本中的HTLV-I，并进行精确定量。
本发明的实施方式并不限于上述实施例所述，在不偏离本发明的精神和范围的情况下，本领域普通技术人员可以在形式和细节上对本发明做出各种改变和改进，而这些均被认为落入了本发明的保护范围。
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Claims (12)

  1. 特异性检测样本中HTLV-I前病毒DNA的引物对，所述引物对包括正向引物5'-CCCGACCTACCTATGGATAATGCT-3'(SEQ ID NO:1)和反向引物5'-GGTTTTGTGGCAGTTGGTTAATACA-3'(SEQ ID NO:2)。
  2. 特异性检测样本中HTLV-I前病毒DNA的引物和探针组合，所述引物对包括正向引物和反向引物，其中正向引物是5'-CCCGACCTACCTATGGATAATGCT-3'(SEQ ID NO:1)，所述反向引物是5'-GGTTTTGTGGCAGTTGGTTAATACA-3'(SEQ ID NO:2)；所述探针的序列为5'-CCCTATGGACAATCAACC-3'(SEQ ID NO:3)，该探针的5’端标记报告荧光基团，3’端标记淬灭基团。
  3. 根据权利要求2的引物和探针组合，其中所述荧光探针的5’端标记的报告荧光基团为FAM，3’端标记的淬灭基团为NFQ-MGB或TAMRA。
  4. 根据权利要求1的引物对或根据权利要求2或3的引物和探针组合在检测样本中HTLV-I前病毒DNA中的应用。
  5. 根据权利要求1的引物对或根据权利要求2或3的引物和探针组合在制备检测样本中HTLV-I前病毒DNA的试剂中的应用。
  6. 根据权利要求4或5的应用，其中所述检测是qPCR检测或数字PCR检测。
  7. 特异性检测样本中HTLV-I前病毒DNA的试剂盒，该试剂盒包括根据权利要求1的引物对，或包括根据权利要求2或3的引物和探针组合。
  8. 根据权利要求7的试剂盒，所述试剂盒还包括选自qPCR反应液、无核酸酶高纯水、阳性对照品、和阴性对照品中的任意一种或更多种试剂。
  9. 样本中HTLV-I前病毒DNA的检测方法，包括使用根据权利要求1的引物对，或使用根据权利要求2或3的引物和探针组合，对样本基因组DNA进行qPCR检测，根据qPCR检测结果定性检测或定量检测样本中是否存在HTLV-I前病毒DNA。
  10. 样本中HTLV-I前病毒DNA的检测方法，包括以下步骤：
    (1)提取样本基因组DNA；
    (2)使用根据权利要求1的引物对，或使用根据权利要求2或3的引物和探针组合，对DNA标准品和样本基因组DNA进行qPCR检测，其中DNA标准品是用具有HTLV-I前病毒DNA的基因组DNA配制的不同给定浓度的样品；
    (3)用DNA标准品的qPCR检测结果制作标准曲线，拟合线性方程，其中R2≥0.99；且各标准品浓度的准确度(RE％)为-75％～150％，确定可稳定检测到的浓度最低点；
    (4)结果判定：如果样本基因组DNA的qPCR结果中出现明显的扩增曲线；且样本基因组DNA的qPCR的Ct值小于浓度最低点的Ct值，则为阳性结果，即样本中存在HTLV-I前病毒DNA；如果无明显扩增曲线，或者有明显扩增曲线，但Ct值大于标准曲线浓度最低点的Ct值，则为阴性结果，即样本中不存在HTLV-I前病毒DNA。
  11. 根据权利要求10的方法，其中步骤(4)进一步包括：根据样本基因组DNA的Ct值和拟合的标准曲线，确定样本基因组DNA中具有HTLV-I前病毒DNA的基因组DNA的浓度。
  12. 样本中HTLV-I前病毒DNA的检测方法，包括以下步骤：
    (1)提取样本基因组DNA；
    (2)使用根据权利要求1的引物对，或使用根据权利要求2或3的引物和探针组合，对DNA标准品和样本基因组DNA进行qPCR检测，其中DNA标准品是用具有HTLV-I前病毒DNA的基因组DNA配制的不同给定浓度的样品；
    (3)用DNA标准品的qPCR检测结果制作标准曲线，拟合线性方程，其中R 2≥0.99；且各标准品浓度的准确度(RE％)为-75％～150％，确定可稳定检测到的浓度最低点；
    (4)结果判定：根据样本基因组DNA的Ct值和拟合的标准曲线，确定样本基因组DNA中具有HTLV-I前病毒DNA的基因组DNA的浓度。
PCT/CN2019/118518 2019-09-19 2019-11-14 特异性检测样本中htlv-i前病毒dna的引物及其应用 WO2021051617A1 (zh)

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