WO2021254109A1 - Hiv-1病毒载量实时荧光定量pcr检测特异性引物对、试剂盒 - Google Patents

Hiv-1病毒载量实时荧光定量pcr检测特异性引物对、试剂盒 Download PDF

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WO2021254109A1
WO2021254109A1 PCT/CN2021/095779 CN2021095779W WO2021254109A1 WO 2021254109 A1 WO2021254109 A1 WO 2021254109A1 CN 2021095779 W CN2021095779 W CN 2021095779W WO 2021254109 A1 WO2021254109 A1 WO 2021254109A1
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hiv
primer
primer pair
viral load
test
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于斌
姜淼
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北京良芯生物科技发展有限公司
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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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    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

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  • the present invention relates to the field of biomolecule detection, in particular to specific primer pairs and kits for real-time fluorescent quantitative PCR detection of HIV-1 viral load.
  • HIV Human Immunodeficiency Virus
  • AIDS Acquired Immunodeficiency Syndrome
  • HIV-2 HIV-1 has four different groups (M, N, O, and P), and each group is caused by independent cross-species transmission. HIV-1 has high virulence and is easier to spread. It also causes most HIV infections in the world.
  • subtype C accounts for 46.6%.
  • B subtype caused 12.1% of infections, followed by A subtype, CRF02_AG, CRF01_AE, G subtype, and D subtype.
  • the F, H, J, and K subtypes together accounted for 0.9% of infections.
  • Other popular recombination types (CRFs) accounted for 3.7%.
  • the change of viral load after HIV-1 infection is closely related to the progression of AIDS.
  • the test of HIV-1 viral load can predict the occurrence, development and prognosis of AIDS.
  • the role of anti-HIV-1 virus drugs is mainly to inhibit the replication of the virus and reduce the viral load. Therefore, if the anti-HIV drugs are effective, the amount of virus in the infected person will decrease. Therefore, the detection of viral load is of great significance for disease monitoring, prevention of mother-to-child transmission, and observation of the efficacy of antiviral drugs.
  • nucleic acid detection has become the development direction of HIV laboratory diagnosis.
  • nucleic acid detection technology has developed rapidly, mainly using various amplification techniques to improve the sensitivity of detection.
  • low-copy target sequences can be amplified and amplified in logarithmic scales.
  • non-radioactive detection systems such as electrochemiluminescence systems
  • Sensitivity, and reduce the pollution of radioactive materials the fluorescent quantitative RT-PCR method based on SYBR Green I fluorescent staining technology has developed into a mature, commonly used and effective detection method.
  • SYBR Green I is a dye with green excitation wavelength that binds to all dsDNA double helix minor groove regions, and does not bind to single-stranded DNA strands. In the free state, SYBR Green I emits weak fluorescence, and once it binds to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, in the PCR system, with the exponential amplification of specific PCR products, the dye is incorporated into the double-stranded DNA during the extension phase of each cycle, and its fluorescence signal intensity is positively correlated with the number of PCR products, which can be detected based on the fluorescence signal Calculate the number of double-stranded DNA in the PCR system.
  • the maximum absorption wavelength of SYBR Green I is about 497nm, and the maximum emission wavelength is about 520nm.
  • the advantages of the SYBR Green I fluorescent dye method are: it can be used to monitor the amplification of any double-stranded DNA sequence. The signal-to-noise ratio is high, the sample fluorescence signal is strong, and the background signal is low; it is easy to use and does not affect the action of other modified enzymes; it is cheap and does not require probes, which reduces the detection setup and operating costs.
  • the purpose of this application is to provide specific primer pairs for real-time fluorescent quantitative PCR detection of HIV-1 viral load.
  • Another object of the present invention is to provide a real-time fluorescent quantitative PCR detection kit for HIV-1 viral load.
  • the embodiment of the present invention provides a specific primer pair for real-time fluorescent quantitative PCR detection of HIV-1 viral load.
  • the primer pair includes primer pair 1 and primer pair 2, wherein primer pair 1 includes the following primers: primer 1: 5'AGTGGGGGGACAYCARGCAGC3 '(SEQ ID No: 1), primer 2: 5'TACTAGTAGTTCCTGCTATRTCACTTCC3' (SEQ ID No: 2); primer pair 2 includes the following primers: Primer 3: 5'GACAGCAGAGAYCCAMTTTGG3' (SEQ ID No: 3), primer 4: 5 'TGCCCCTTCACCTTTCCA3' (SEQ ID No: 4).
  • the embodiment of the present invention provides a real-time fluorescent quantitative PCR detection kit for HIV-1 viral load.
  • the kit includes primer pair 1 and primer pair 2, wherein primer pair 1 includes the following primers: primer 1: 5'AGTGGGGGGACAYCARGCAGC3', Primer 2: 5'TACTAGTAGTTCCTGCTATRTCACTTCC3'; primer pair 2 includes the following primers: primer 3: 5'GACAGCAGAGAYCCAMTTTGG3', primer 4: 5'TGCCCCTTCACCTTTCCA3'.
  • the human immunodeficiency virus type I (HIV-1) RNA can be detected by one-step fluorescence quantitative RT-PCR technology, so the operation is simple, the detection time is shortened, and the pollution is reduced.
  • the primer pair of the present invention has high specificity, high sensitivity and good repeatability, and the detection threshold of the sensitivity and specificity of the primer is 4 ⁇ 10 1 to 1 ⁇ 10 8 copies/mL.
  • the primer design of the present invention is based on the positions of two HIV genes, the positions of gag and pol, which can more accurately reflect the true viral load level.
  • Figure 1 is an amplification curve when primer pair 1 is used for amplification according to a specific embodiment of the present invention, where ⁇ Rn represents the increase in fluorescence, that is, the increase in amplified product, and the Ct value represents the number of cycles;
  • Figure 2 is a standard curve amplified by primer pair 1 according to a specific embodiment of the present invention, wherein the abscissa represents the logarithmic value of the viral load, and the ordinate represents the Ct value, which represents the number of cycles;
  • Fig. 3 is an amplification curve when a standard product is amplified using primer pair 2 according to a specific embodiment of the present invention, where ⁇ Rn represents the increase in fluorescence, that is, the increase in amplification product, and the Ct value represents the number of cycles;
  • Fig. 4 is a standard curve amplified by primer pair 2 according to a specific embodiment of the present invention, wherein the abscissa represents the logarithmic value of the viral load, and the ordinate represents the Ct value, which represents the number of cycles;
  • Figure 5 shows the comparison of the results of testing the viral load of 16 samples between the present invention and similar products in the market
  • Figure 6 shows the results of the primer sensitivity verification test, where ⁇ Rn represents the increase in fluorescence, that is, the increase in amplification product, and the Ct value represents the number of cycles;
  • Figure 7 is the first test result of the primer stability verification test, where ⁇ Rn represents the increase in fluorescence, that is, the increase in amplification product, and the Ct value represents the number of cycles;
  • Figure 8 is the second test result of the primer stability verification test, where ⁇ Rn represents the increase in fluorescence, that is, the increase in amplification product, and the Ct value represents the number of cycles;
  • Figure 9 is the third test result of the primer stability verification test, where ⁇ Rn represents the increase in fluorescence, that is, the increase in amplification product, and the Ct value represents the number of cycles;
  • Figure 10 shows the comparison between the optimized primers and similar products on the market to detect the viral load of 16 samples.
  • two pairs of specific primers are used to detect human immunodeficiency virus type I (HIV-1) RNA using one-step real-time fluorescent quantitative PCR technology.
  • Two amplification standard curves are obtained, and the accuracy of viral load detection is further improved by data analysis of these two standard curves.
  • the reaction system is directly prepared for amplification. The reaction system is easy to prepare, the amplification procedure is simple, the amplification time is short, and there is no need to repeat the cycle multiple times to prevent product contamination.
  • the principle of the method of the present invention is based on the principle of SYBR Green I real-time fluorescent quantitative PCR, using HIV-1 RNA as a template, using two pairs of viral genome-specific primers, plus reverse transcriptase, Taq-DNA polymerase, and one-step fluorescent RT -PCR experiment to realize rapid and accurate analysis of HIV-1 RNA template. From reverse transcription to real-time fluorescent quantitative PCR, it can be completed in one step, which can effectively prevent multi-step operation contamination. Therefore, the detection method and kit of the present invention have high accuracy, strong specificity, high sensitivity, simple operation, clear results, and high reliability, and can be used for the quantitative detection of HIV-1 in serum.
  • the present invention selects two separate locations on the HIV-1 genome for amplification and detection.
  • the data is obtained by calculating the weighted average of the two load data, which is more specific and more realistic than using single-site fluorescent PCR amplification. Reflects the HIV load level in the body.
  • the method of using the kit of the present invention is as follows.
  • reaction procedure is: reaction at 48°C for 30 minutes, reaction at 95°C for 10 seconds, then press 95°C for 15s ⁇ 57°C 30s ⁇ 72°C for 1min, cycle 40 times, and collect the FAM fluorescence channel signal at 72°C, and then pass 95°C for 15s, 60 °C 30s, 95°C 1s to get the melting curve.
  • the results are saved and data analyzed according to the requirements of the instrument and software. Taking the fluorescence value higher than the noise line of the sample and the negative control as the detection threshold, the analysis software automatically combined with the standard curve to calculate the HIV-1 RNA content C (copies/mL) of each sample extract.
  • the detection kit includes: an RT-PCR reaction solution, an RT-PCR enzyme mixture, primers, a negative control, and calibrators No. 1 to No. 5.
  • the enzyme mixture is a mixture of reverse transcriptase (3U/ ⁇ L) and TaqDNA polymerase (2U/ ⁇ L);
  • the primers provided are two pairs of HIV-1 specific primers (each primer is 1 ⁇ M);
  • the negative control is normal human serum without HIV-1 RNA;
  • the calibrator No. 1 to No. 5 provided are those with a known concentration gradient Plasmid of HIV-1 gene
  • the human immunodeficiency virus type I one-step fluorescence quantitative PCR detection kit was used to detect RNA extracted from 16 HIV-1 positive serum samples.
  • the results are saved and data analyzed according to the requirements of the instrument and software. Taking the fluorescence value higher than the noise line of the sample and the negative control as the detection threshold, the analysis software automatically combined the two standard curves to calculate the HIV-1 RNA content C (copies/mL) of each sample extract, and the average value was taken as the final result. Then analyze the melting curve to ensure that there is no primer dimer interference in the PCR process.
  • the amplification curve and standard curve of the standard amplified by primer pair 1 are shown in Figure 1-2, respectively.
  • the amplification curve and standard curve of the standard amplified by primer pair 2 are shown in Figure 3-4, respectively.
  • the standard curves R 2 are all greater than 0.99, and the Ct values of the samples are all less than 35, which meets the requirements.
  • the analysis software automatically combined the two standard curves to calculate the HIV-1 RNA content C (copies/mL) of each sample extract. The results are shown in Table 1.
  • Viral load unit (copies/mL)
  • the amplification results of primer pair 1 and primer pair 2 are automatically given by QuantStudio TM Design & Analysis SE Software. Take the average of the two as the final viral load result, and compare it with the viral load result detected by similar products on the market (as shown in Figure 5). The results of the two are similar, which proves that the kit of the present invention is effective on the 16 samples tested.
  • the viral load of HIV-1 has a good detection effect.
  • the samples were first sequenced, and the obtained sequences were submitted to the Stanford University HIV Drug Resistance Database (https://hivdb.stanford.edu/hivdb) for subtype analysis. Afterwards, the kit provided by the present invention was used to detect the viral load of each sample (the method and procedure are the same as in Example 1), and the results are shown below.
  • Sensitivity verification test The template DNA is serially diluted in a 10-fold gradient, and 5 ⁇ L of each order of dilution is taken as the amplification template from 5.92 ⁇ 10 3 to 5.92 ⁇ 10 9 copies/mL.
  • the test conditions are the same as in Example 1.
  • Stability verification test aliquot three sets of primers and standard products provided by the present invention at one time, and store them at -20°C. On the 0th, 3rd, and 6th day, the RT-PCR amplification test was performed (the test method and procedure were the same as in Example 1), and the Ct values of the same group of standard products were compared with three RT-PCR to judge the stability of the primers. The results of the three tests are shown in Figure 7, Figure 8, and Figure 9. The statistical results of the Ct value and the coefficient of variation are shown in Table 2 below.
  • the coefficient of variation of the Ct value of each group is less than 1%, which shows that the primers have good stability.
  • Viral load unit copies/mL

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Abstract

本发明公开了HIV-1病毒载量实时荧光定量PCR检测特异性引物对和试剂盒，利用一步法荧光定量RT-PCR技术检测HIV-1病毒载量。

Description

HIV-1病毒载量实时荧光定量PCR检测特异性引物对、试剂盒
交叉引用
本发明要求在2020年06月18日提交中国专利局、申请号为202010558549.8、发明名称为“HIV-1病毒载量实时荧光定量PCR检测特异性引物对、试剂盒”的中国专利申请的优先权，该申请的全部内容通过引用结合在本发明中。
技术领域
本发明涉及生物分子检测领域，具体涉及HIV-1病毒载量实时荧光定量PCR检测特异性引物对、试剂盒。
背景技术
人类免疫缺陷病毒(Human Immunodeficiency Virus,HIV)是诱发人获得性免疫缺陷综合征(Acquired Immunodeficiency Syndrome,AIDS)的主要病原体，在分类学上属于逆转录病毒科(Retroviridae)慢病毒属(Lentivirus)。根据基因型的不同，HIV可以分为两种类型：HIV-1与HIV-2。HIV-1有M、N、O、P四个不同的组(group)，每个组由独立跨物种传播所致。HIV-1具有较高的毒力，更容易传播的同时也导致了全球绝大多数HIV感染。
全球所有HIV-1感染中，C亚型占46.6％。B亚型导致12.1％的感染，其次是A亚型、CRF02_AG、CRF01_AE、G亚型、D亚型。F、H、J、K亚型合计占感染的0.9％。其他流行重组类型(CRFs)占3.7％。
HIV-1感染后病毒载量的变化与艾滋病的发病进程有密切的相关性，HIV-1病毒载量的检测可以预测艾滋病发生、发展以及预后。抗HIV-1病毒药物的作用主要是抑制病毒的复制，降低病毒载量，因此，抗HIV的药物若产生效果，感染者体内的病毒量就会下降。因此，病毒载量的检测对疾病的 监控、预防母婴传播以及观察抗病毒药物的疗效都具有重要的意义。
核酸检测已经成为了HIV实验室诊断的发展方向，最近几年核酸检测技术发展迅速，主要是采用各种扩增放大技术提高检测的灵敏度。随着扩增技术的成熟，可以将低拷贝的靶序列成对数级放大扩增，同时采用比放射性探针更灵敏的非放射性检测系统(如电化学发光系统等)，明显提高核酸检测的灵敏度，并减少放射性物质的污染，基于SYBR Green Ⅰ荧光染色技术的荧光定量RT-PCR方法已发展成为成熟、常用而有效的检测手段。
SYBR Green I是一种结合于所有dsDNA双螺旋小沟区域的具有绿色激发波长的染料，并不与单链DNA链结合。在游离状态下，SYBR Green I发出微弱的荧光，一旦与双链DNA结合后，荧光大大增强。因此，在PCR体系中，随着特异性PCR产物的指数扩增，每个循环的延伸阶段，染料掺入双链DNA中，其荧光信号强度与PCR产物的数量呈正相关，可以根据荧光信号检测出PCR体系存在的双链DNA数量。SYBR Green I的最大吸收波长约为497nm，发射波长最大约为520nm。SYBR Green I荧光染料法的优点有：可用于监测任何双链DNA序列的扩增。信噪比高，样品荧光信号强，背景信号低；使用方便，不影响其它修饰酶作用；价格便宜，无需探针，降低了检测的设置和运行成本。
虽然HIV-1病毒各亚型的序列已公开，但是针对于个体感染人，序列的变化还是非常大的，因此仅基于已经公开的数据资料获得的HIV-1病毒的保守序列所设计的引物难以广泛适用，尤其不适于检测中国特有的HIV-1感染者。
发明内容
为了解决上述问题提出并完成本发明。
本申请的目的是提供HIV-1病毒载量实时荧光定量PCR检测特异性引物对。
本发明的再一目的是提供HIV-1病毒载量实时荧光定量PCR检测试剂盒。
本发明实施例提供了HIV-1病毒载量实时荧光定量PCR检测特异性引物对，所述引物对包括引物对1和引物对2，其中，引物对1包括以下引物：引物1：5’AGTGGGGGGACAYCARGCAGC3’(SEQ ID No:1)，引物2：5’TACTAGTAGTTCCTGCTATRTCACTTCC3’(SEQ ID No:2)；引物对2包括以下引物：引物3：5’GACAGCAGAGAYCCAMTTTGG3’(SEQ ID No:3)，引物4：5’TGCCCCTTCACCTTTCCA3’(SEQ ID No:4)。
本发明实施例提供了HIV-1病毒载量实时荧光定量PCR检测试剂盒，所述试剂盒包括引物对1和引物对2，其中，引物对1包括以下引物：引物1：5’AGTGGGGGGACAYCARGCAGC3’，引物2：5’TACTAGTAGTTCCTGCTATRTCACTTCC3’；引物对2包括以下引物：引物3：5’GACAGCAGAGAYCCAMTTTGG3’，引物4：5’TGCCCCTTCACCTTTCCA3’。
本发明的技术方案的有益效果：
1、利用本发明的引物对，可以通过一步法荧光定量RT-PCR技术，检测人类免疫缺陷病毒Ⅰ型(HIV-1)RNA，因此操作简单，缩短检测时间，减少了污染。
2、本发明的引物对具有高特异性、高敏感性和良好的重复性，引物的灵敏性和特异性的检测阈值为4×10 1～1×10 8copies/mL。
3、可用于HIV-1定量检测，并且作为临床对HIV-1感染的辅助诊断方法和临床治疗效果的监测手段。
4、本发明的引物设计是基于两个HIV的基因位置，gag和pol的位置，可以更加精确的反映出真实的病毒载量水平。
附图说明
此处所说明的附图用来提供对本申请的进一步理解，构成本申请的一部分，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。在附图中：
图1为根据本发明的具体实施例利用引物对1进行扩增时的扩增曲线，其中，ΔRn代表荧光增量，亦即扩增产物增量，Ct值表示循环数；
图2是根据本发明的具体实施例利用引物对1扩增的标准曲线，其中，横坐标代表病毒载量的对数值，纵坐标代表Ct值，表示循环数；
图3为根据本发明的具体实施例标准品利用引物对2进行扩增时的扩增曲线，其中，ΔRn代表荧光增量，亦即扩增产物增量，Ct值表示循环数；
图4是根据本发明的具体实施例利用引物对2扩增的标准曲线，其中，横坐标代表病毒载量的对数值，纵坐标代表Ct值，表示循环数；
图5显示本发明与市场同类产品检测16个样品病毒载量的结果对比；
图6为引物灵敏性验证试验结果，其中，ΔRn代表荧光增量，亦即扩增产物增量，Ct值表示循环数；
图7为引物稳定性验证试验第一次试验结果，其中，ΔRn代表荧光增量，亦即扩增产物增量，Ct值表示循环数；
图8为引物稳定性验证试验第二次试验结果，其中，ΔRn代表荧光增量，亦即扩增产物增量，Ct值表示循环数；
图9为引物稳定性验证试验第三次试验结果，其中，ΔRn代表荧光增量，亦即扩增产物增量，Ct值表示循环数；
图10为显示优化引物与市场同类产品检测16个样品病毒载量的结果对比。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚，下面将结合本申请具体实施例及相应的附图对本申请技术方案进行清楚、完整地描述。显然，所描 述的实施例仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
以下结合附图，详细说明本申请各实施例提供的技术方案。
根据本发明的具体实施方式，利用两对特异性引物采用一步法实时荧光定量PCR技术对人类免疫缺陷病毒Ⅰ型(HIV-1)RNA进行检测。得到两条扩增标准曲线，通过对这两组标准曲线进行数据分析，进一步提高病毒载量检测精准度。在对HIV-1阳性血清样本进行核酸提取后，直接配制反应体系进行扩增，反应体系配制方便，扩增程序步骤简便，扩增时间短，无需多次重复循环、防止产物污染。
本发明方法原理是基于SYBR Green Ⅰ实时荧光定量PCR原理，以HIV-1 RNA为模板，分别利用两对病毒基因组特异性引物，加之以逆转录酶、Taq-DNA聚合酶，经一步法荧光RT-PCR实验，实现快速、准确地对HIV-1 RNA模板进行分析。从逆转录到实时荧光定量PCR，一步即可完成，可有效的防止多步骤操作污染。所以，本发明的检测方法及试剂盒精确度高、特异性强、敏感度高、操作简便、结果明晰、可靠性高，可用于血清中HIV-1的定量检测。
此外本发明分别选用HIV-1基因组上分开的两个位置扩增检测，通过计算两个载量数据的加权平均得到数据，比采用单一位点的荧光PCR扩增特异性更强，更加能够真实的反映出体内HIV的载量水平。
使用本发明试剂盒使用方法如下。
每次检测均应设立阴性对照和校准品1号～5号；按反应样本数n(反应样本数＝待检样品数+对照品1个+校准品5个+1)配制反应液；取RT-PCR反应液n×10.0μL、引物n×2μL、RT-PCR酶混合物n×3μL于一离心管中混匀，低速离心数秒，按15μL/管分装到反应管中；取样本提取物、阴性对照提取物、校准品提取物各5μL分别加入反应管中，低速离心数秒，取 出置全自动荧光定量PCR仪上。
反应程序为：48℃反应30min，95℃反应10s，然后按95℃ 15s→57℃ 30s→72℃ 1min，循环40次，并于72℃采集FAM荧光通道的信号，之后经95℃ 15s，60℃ 30s，95℃ 1s得到熔解曲线。
仪器PCR程序运行完成后按仪器及软件要求进行结果保存和数据分析。以取高于样本噪声线和阴性对照的荧光值作为检测阈值，分析软件自动结合标准曲线计算各样品提取物的HIV-1 RNA含量C(copies/mL)。
根据本发明的具体实施方式，检测试剂盒包括：RT-PCR反应液、RT-PCR酶混合物、引物、阴性对照、校准品1号～5号。对提取好的HIV-1 RNA模板直接进行实时荧光定量RT-PCR检测，其中，所述的RT-PCR反应液为Tris-HCl(pH＝8.3)20mM、KCl 100mM、明胶0.2mg/ml、dATP、dGTP、dCTP、dUTP各0.4mM、MgCl 2 6mM、SYBR Green Ⅰ 2×的混合液；所述的酶混合物为逆转录酶(3U/μL)、TaqDNA聚合酶(2U/μL)的混合物；所提供的引物为两对HIV-1特异性引物(引物各1μM)；所提供的阴性对照为无HIV-1 RNA的正常人血清；所提供校准品1号～5号为已知浓度梯度的含有HIV-1基因片段的质粒。
实施例1
利用人类免疫缺陷病毒Ⅰ型一步法荧光定量PCR检测试剂盒对从16份HIV-1阳性血清样本提取的RNA进行检测。
利用Applied Biosystems QuantStudio5 Real-Time PCR Systems进行实时荧光定量PCR，于72℃收集FAM通道荧光信号，循环过程结束后进行熔解曲线步骤。
仪器RT-PCR程序运行完成后按仪器及软件要求进行结果保存和数据分析。以取高于样本噪声线和阴性对照的荧光值作为检测阈值,分析软件自动结合两条标准曲线计算各样品提取物的HIV-1 RNA含量C(copies/mL),取平均值作为最终结果。之后分析熔解曲线，确保PCR过程无引物二聚体干扰。
利用引物对1扩增标准品的扩增曲线和标准曲线分别如图1-2所示。利用引物对2扩增标准品的扩增曲线和标准曲线分别如图3-4所示。
标准曲线R 2均大于0.99，且样品Ct值均小于35，符合要求。分析软件自动结合两条标准曲线计算各样品提取物的HIV-1 RNA含量C(copies/mL)，结果如下表1。
表1
Figure PCTCN2021095779-appb-000001
Figure PCTCN2021095779-appb-000002
注：病毒载量单位：(copies/mL)
如表1所示，引物对1和引物对2的扩增结果由QuantStudio TM Design&Analysis SE Software自动给出。取二者平均值作为最终的病毒载量结果，并与利用市场同类产品检测到的病毒载量结果进行比较(如图5)，二者结果相近，证明本发明试剂盒对所测16个样本HIV-1的病毒载量具有良好的检测效果。
实施例2、对各种亚型HIV-1的检测结果
对样本进行一代测序，并将所得序列提交到斯坦福大学HIV耐药数据库(https://hivdb.stanford.edu/hivdb)进行亚型分析。之后利用本发明提供的试剂盒检测各个样本的病毒载量(方法及程序同实施例1)，结果如下所示。
(1)样本编号R16，测序结果(SEQ ID No:5)。
HIV-1亚型为B；扩增结果；Ct＝26.681
(2)样本编号T1516，测序结果(SEQ ID No:6)。
HIV-1亚型为CRF01_AE；扩增结果：Ct＝23.259
(3)样本编号：Y4，测序结果(SEQ ID No:7)。
HIV-1亚型为CRF07_BC；扩增结果：Ct＝27.806
(4)样本编号：Y10，测序结果(SEQ ID No:8)。
HIV-1亚型为B+C；扩增结果：Ct＝26.935
在检测中国常见的4种亚型的HIV-1病毒载量试验中，实验结果Ct值均小于35，证明本发明提供的引物对对各种亚型HIV-1均具有良好的检测效果。
实施例3、灵敏性和稳定性验证试验。
1.灵敏性验证试验：将模板DNA以10倍梯度进行系列稀释,从5.92×10 3～5.92×10 9copies/mL区间取每一数量级的稀释液5μL作为扩增模板。 试验条件同实例1。结果显示：在Ct＝30处可见到明显的扩增曲线(见图6)，证明能够检测到的最低稀释度<5.92×10 3copies/mL(大约相当于检测最低限度为每个反应30个DNA拷贝量)，具有良好的灵敏度，每个反应都可最低检测到30个拷贝数DNA。
2.稳定性验证试验：一次分装三组本发明提供的引物和标准品，于-20℃贮存。在第0、3、6日分别进行RT-PCR扩增试验(试验方法和程序同实施例1)，比较同一组标准品三次RT-PCR的Ct值，判断引物稳定性。三次试验结果见图7、图8、图9，Ct值统计结果和变异系数如下表2所示。
表2
第一次PCR 第二次PCR 第三次PCR 平均Ct值 变异系数(CV)
11.017 11.193 11.189 11.133 0.74％
13.889 14.046 14.145 14.02667 0.75％
16.939 16.939 17.116 16.998 0.49％
19.926 20.116 20.353 20.13167 0.87％
23.668 23.992 24.204 23.95467 0.92％
分析统计结果，每组Ct值的变异系数均小于1％，可知引物具有良好的稳定性。
利用一对未优化过的引物(序列为：F1＝5’GGCAGCAATGCAAATGTTAAA R1＝TCATCTGGCCTGGTGGAATA3’)，检测同一组16个样本的病毒载量(验证方法及程序同实施例1)，结果如下表3所示。
表3
样本序号 载量值 市场同类产品检测结果 变异系数(CV)
1 10694 255925 91.98％
3 14583 52183 56.32％
4 97641 155173 22.76％
5 35719 157315 62.99％
6 13647 166182 84.82％
7 65072 77126 8.48％
8 104859 307952 49.20％
9 33671 107908 52.44％
10 197425 291521 19.24％
11 79853 168476 35.69％
12 16594 141945 79.07％
13 68504 79269 7.28％
14 47203 72512 21.14％
15 12541 291521 91.75％
16 15371 186718 84.79％
注：病毒载量单位：copies/mL
如表2和图10所示，比较该对引物和市场同类产品检测病毒载量的结果，二者检测结果变异系数很大，可见此对引物检测病毒载量的能力不如经过优化的引物对。
以上所述仅为本申请的实施例而已，并不用于限制本申请。对于本领域技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原理之内所作的任何修改、等同替换、改进等，均应包含在本申请的权利要求范围之内。

Claims (3)

  1. HIV-1病毒载量实时荧光定量PCR检测特异性引物对，其中，所述引物对包括引物对1和引物对2，其中，
    引物对1包括以下引物：
    引物1：5’AGTGGGGGGACAYCARGCAGC3’，
    引物2：5’TACTAGTAGTTCCTGCTATRTCACTTCC3’；
    引物对2包括以下引物：
    引物3：5’GACAGCAGAGAYCCAMTTTGG3’
    引物4：5’TGCCCCTTCACCTTTCCA3’。
  2. HIV-1病毒载量实时荧光定量PCR检测试剂盒，其中，所述试剂盒包括引物对1和引物对2，其中，
    引物对1包括以下引物：
    引物1：5’AGTGGGGGGACAYCARGCAGC3’，
    引物2：5’TACTAGTAGTTCCTGCTATRTCACTTCC3’；
    引物对2包括以下引物：
    引物3：5’GACAGCAGAGAYCCAMTTTGG3’，
    引物4：5’TGCCCCTTCACCTTTCCA3’。
  3. 权利要求1所述的HIV-1病毒载量实时荧光定量PCR检测特异性引物对用于制备鉴定HIV-1病毒载量的试剂的应用。
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