CN1094154C - A fluorescent quantitative polymerase chain reaction method and kit - Google Patents

A fluorescent quantitative polymerase chain reaction method and kit Download PDF

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CN1094154C
CN1094154C CN 99100669 CN99100669A CN1094154C CN 1094154 C CN1094154 C CN 1094154C CN 99100669 CN99100669 CN 99100669 CN 99100669 A CN99100669 A CN 99100669A CN 1094154 C CN1094154 C CN 1094154C
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nucleic acid
primer
pcr
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程钢
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中山医科大学达安基因股份有限公司
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本发明提供了一种荧光定量PCR方法。 The present invention provides a method of quantitative PCR. 该方法的荧光PCR反应体系中镁离子的浓度为15-20mM,Taq酶用量为8-10单位/反应;在PCR反应前测定反应前荧光值;在PCR反应后测得反应后荧光值;计算出差值。 Fluorescence PCR reaction system of the method of the magnesium ion concentration is 15-20mM, Taq enzyme concentration of 8-10 units / reaction; fluorescence measured prior to the reaction prior to the PCR reaction; after the reaction was measured after the PCR reaction fluorescence value; calculated travel value. 同时用系列阳性和阴性模板也进行同样的步骤,制成标准曲线;用各样品的荧光增值即可在标准曲线上查出相应的样品中的核酸起始拷贝数。 Meanwhile series with positive and negative template subjected to the same procedure to prepare the standard curve; can be isolated starting copy number of nucleic acid in the respective sample standard curve for each sample using the fluorescence value. 本发明还提供了一种用于该方法的试剂盒。 The present invention also provides a kit for the method.

Description

一种荧光定量聚合酶链式反应方法及其试剂盒 A fluorescent quantitative polymerase chain reaction method and kit

本发明涉及用于核酸检测的PCR(polymerase chain reaction,聚合酶链式反应)以及用于该方法的试剂盒。 The present invention relates to nucleic acid detection for PCR (polymerase chain reaction, polymerase chain reaction) as well as a kit for the method. 具体地说,本发明涉及用于核酸检测的荧光定量PCR方法及其试剂盒。 More specifically, the present invention relates to a quantitative PCR method and kit for nucleic acid detection.

聚合酶链式反应(Polymerase Chain Reaction,PCR)是基于DNA体外扩增技术来检测痕量、微量核酸的方法,已被广泛用于核酸的检测分析和遗传学测试[参见Bevan et.al,PCR methods andApplications,1:222-228,(1992)]。 Polymerase chain reaction (Polymerase Chain Reaction, PCR) is an in vitro DNA amplification technique based on the detection of trace, trace amounts of nucleic acids, it has been widely used to detect genetic testing and analysis of nucleic acids [see Bevan et.al, PCR methods andApplications, 1: 222-228, (1992)]. 通过PCR过程,可以准确检出临床样本中与疾病相关的痕量DNA/RNA的存在,明确检查出致病原因,从而指导临床诊断和治疗。 By PCR process can be accurately detected in the presence of a disease associated with clinical samples trace DNA / RNA, a clear cause of check out, so clinical diagnosis and treatment. 进一步的定量PCR结果,则可以更详细的监测临床样本中与疾病相关的痕量DNA/RNA的数量及其变化,对于临床治疗方案的选择、疗效考核有极大的指导意义,尤其适合许多源于DNA/RNA数量变化的疾病的检测和治疗。 Further quantitative PCR results, it can be more detailed monitoring of clinical specimens associated with disease traces of DNA / RNA and the number of changes to clinical treatment, the efficacy assessment has great significance, especially for the many sources in DNA / detection and treatment of diseases of varying the number of RNA.

PCR方法采用一对人工合成的寡聚核苷酸引物及dNTPs为原料,在体外模拟DNA的体外复制过程,用耐高温的DNA聚合酶经多次循环,扩增模板DNA。 PCR methods using a pair of synthetic oligonucleotide primers and dNTPs as raw materials vitro DNA replication in vitro process, high temperature resistant DNA polymerase after multiple cycles, amplification of template DNA. 可以将目的DNA的量扩增106~107倍,再将产物在琼脂糖凝胶上电泳,以溴乙锭(EB)染色体而显示结果。 The amount of target DNA can be amplified 106 to 107 times, and the product on agarose gel electrophoresis to ethidium bromide (EB) shows the results of chromosome. 此方法具有灵敏、迅速、准确、方便等特点。 This method is sensitive, rapid, accurate, and convenient. 但它也有相应的不足,主要可归结为3点:(1)只有定性结果,无法给出临床需要的定量结果;(2)由于采用电泳检测,易引起PCR产物的交叉污染,增加了假阳性结果的可能性;(3)采用的染色剂溴乙锭是强烈致癌物,可能危害操作人员及污染环境。 But it also has a corresponding shortage, it can mainly be attributed to three points: (1) only qualitative results, not give quantitative results of clinical needs; (2) As a result of electrophoresis, can lead to cross-contamination of PCR products, an increase of false positives the results of the likelihood; (3) ethidium bromide staining agent used is a strong carcinogen, could endanger the operator and environmental pollution.

经过研究和改进常规PCR方法,又产生了实时定量荧光PCR方法(参见Kenneth J.Lival et al,US patent 5,538,848)。 After research and improvement conventional PCR method, and it generates a real-time quantitative fluorescence PCR methods (see, Kenneth J.Lival et al, US patent 5,538,848). 该技术在常规PCR基础上,添加了一条荧光标记的探针。 The conventional technique based on PCR, adding a fluorescently labeled probe. 探针在无特异性PCR发生时,荧光信号不改变,当有特异性PCR扩增发生时,探针会在PCR过程中被切断而引起荧光信号的增长(见图1)。 When no specific PCR probes and the fluorescence signal does not change, when a specific PCR amplification occurs, the probe will be cut off during the PCR fluorescence signal caused by growth (see FIG. 1). 荧光信号伴随着PCR的过程的进行,PCR产物的增长而增长。 With the fluorescence signal for the PCR process, a PCR product growth and growth. 实时定量荧光PCR方法就是利用此原理,在PCR过程中,连续不断地检测反应体系中的荧光信号的变化。 Real-time quantitative fluorescence PCR method is to use this principle, in the PCR process, continuously detecting a change in the fluorescent signal of the reaction system. 当信号增强到某一阈值时,此时的循环次数(Ct值)就被记录下来,该循环参数和PCR体系中起始DNA量的对数值之间有严格的线性关系[参见Higuchi etal,Biotechkology,11:1026-1030(1993)],根据循环参数Ct值就可以准确定出起始DNA的数量。 When the signal is enhanced to a certain threshold, then the number of cycles (Ct value) was recorded, the cycle number and the amount of starting DNA PCR system of strict linear relationship between the value [see Higuchi etal, Biotechkology , 11: 1026-1030 (1993)], according to the cycle parameters Ct value can accurately fix the number of starting DNA. 实际使用时,是将含有荧光探针和从标本中提取的核酸样品的PCR反应管放入PCR-荧光检测-电脑分析一体化仪器,启动PCR循环过程和荧光检测功能,后者在仪器中透过样品管盖(Eppendoff管盖)直接实现闭管检测。 Actual use, a PCR reaction containing the fluorescent probe and a nucleic acid sample extracted from the specimen tube into the PCR- fluorescence detection - Integration computer analysis instrument, start PCR cycling and fluorescence detection, through which the instrument through sample tube cap (Eppendoff tube lid) directly implement closed tube detection. 反应结束后,仪器将自动分析检出样品的Ct值;同时平行测定阳性梯度标准品的Ct值,用这些值制成标准曲线,再由样品的Ct值,可在标准曲线上查出相应的起始DNA量。 After completion of the reaction, the instrument will automatically analyze samples Ct values ​​detected; Ct values ​​measured while the positive gradient standard parallel with the standard curve values, then the Ct value of the sample, can be detected on the corresponding standard curve the amount of starting DNA. 本方法由于采用荧光技术和闭管检测,完全克服了前述常规PCR方法的3个主要缺点。 As a result of the present process and the closed tube fluorescence detection, completely overcome three major drawback of the conventional PCR method. 但出于实时连续检测的需要,技术中必须采用昂贵的PCR-荧光检测-电脑分析一体化仪器,如美国Perkin Elmer公司的7700型Sequence Detector,价格高达10万美元。 But for the needs of real-time continuous detection technology must use expensive PCR- fluorescence detection - the integration of computer analysis instrument, such as the US company Perkin Elmer 7700 Sequence Detector, the price of up to $ 100,000. 这种情况限制了实时定量荧光PCR方法在临床诊断中的大规模应用。 This limits large-scale real-time fluorescence quantitative PCR method in clinical diagnosis.

本发明的目的是为了克服上述已有荧光PCR检测方法实施费用高,使用仪器昂贵的缺点,提供一种简便、价廉、准确率高的定量荧光PCR检测方法以及用于该方法的试剂盒。 Object of the present invention is to overcome the above-described PCR assay embodiment have a high cost, expensive instrumentation disadvantages and to provide a simple, inexpensive, high accuracy quantitative PCR assay and a kit for the method.

本发明的荧光PCR方法包括如下步骤:a、根据待检测靶基因的特异性核酸序列合成一对引物。 Fluorescence PCR method of the present invention comprises the steps of: a, using a pair of primers specific nucleic acid sequence to be detected in accordance with the target gene. 引物相互之间最好至少相距30个碱基;b、根据待检测靶基因的特异性核酸序列中位于两个引物之间的序列设计、合成一个荧光探针;c、使用步骤a中的引物和步骤b中的荧光探针及其它PCR成份组成荧光PCR反应体系,其中镁离子的浓度为15-20mM,Taq酶用量为8-10单位/反应;d、从待测标本中提取DNA,或是提取RNA然后逆转录成cDNA,加入步骤c中的反应体系中,经离心混合后测定反应前荧光值; The primer is preferably at least 30 bases away from each other; B, according to the specific nucleic acid sequence to be detected in the target gene sequences located between the two primers were designed, synthesized a fluorescent probe; C, step a using the primers PCR and fluorescent probes and other ingredients in the composition of step b fluorescent PCR reaction system, wherein the magnesium ion concentration is 15-20mM, Taq enzyme concentration of 8-10 units / reaction; D, DNA extracted from the specimen to be tested, or RNA was extracted and reverse transcribed into the cDNA, was added in the reaction system of step c, the fluorescent value measured before the reaction by centrifugation after mixing;

e、将反应管放入PCR仪进行20~50次循环的PCR反应;反应结束后再测定一次反应管中的荧光值;前后两次测值的差值即代表了本管反应的荧光增值;f、同时用系列阳性和阴性模板也进行步骤d的核酸提取和PCR反应,测得各自相应的荧光增值,将荧光增值相对于阳性模板的初始核酸量的自然对数值作图,制成标准曲线;g、用上述e步骤中获得的各样品的荧光增值,可在标准曲线上查出相应的样品中的核酸起始拷贝数。 e, the reaction tube was placed in PCR machine PCR reactions were performed 20 to 50 cycles; fluorescence tube after completion of the reaction the reaction time was measured; the difference between two consecutive measurement value which represents the present value of the fluorescent tube reaction; F, also with a series of positive and negative for the template nucleic acid of step d extraction and PCR reactions, fluorescence respective measured value, the relative value of the fluorescent values ​​plotted to the initial amount of nucleic acid template positive natural, standard curve ; G, with the fluorescence value of each sample obtained in step e, a nucleic acid can be isolated starting copy number of the corresponding sample on the standard curve.

在本发明的方法中,靶基因的特异性核酸序列是指仅存在于该靶基因中的核酸序列。 In the method of the present invention, the specific nucleic acid sequence of the target gene means a nucleic acid sequence present only in the target gene. 该核酸序列的存在与所述靶基因的存在之间是具有一一对应的关系。 Exists between the presence of the nucleic acid sequence of the target gene is a one to one relationship.

本发明方法的PCR反应体系与常规PCR反应体系类似,包括高温聚合酶,核酸提取试剂,荧光PCR反应液,阳性模板系列,阴性模板和覆盖剂等。 PCR reaction system of the process of the present invention is similar to conventional PCR reaction system, including high temperature polymerase, a nucleic acid extraction reagent, fluorescent PCR reaction solution series positive template and negative template and a cover or the like. 参见Bevan等的上述文献。 See Bevan et al above references.

高温聚合酶是指具有3'→5'DNA聚合酶活性、5'→3'DNA外切核酸活性和可耐受高温的聚合酶。 It refers to a high-temperature polymerase 3 '→ 5'DNA polymerase activity, 5' → 3'DNA exonuclease activity of the polymerase and can withstand high temperatures. 所述的高温聚合酶最好是95℃半衰期>45分钟的高温聚合酶。 The polymerase is preferably a high-temperature 95 deg.] C half-life> 45 minutes of high-temperature polymerase.

荧光PCR反应液是含dNTPs,缓冲体系,PCR扩增引物和荧光探针的体系。 Fluorescence PCR reaction solution containing dNTPs, buffer systems, systems PCR amplification primers and fluorescent probes.

所述的PCR扩增引物是经过设计,特异性针对某一待测核酸序列的特征序列的引物,长度15~40碱基(bases),最好是18~25碱基(bases)。 The PCR amplification primers have been designed, the primers specific for signature sequences a sample nucleic acid sequence, the length of 15 ~ 40 bases (bases), preferably 18 to 25 bases (bases).

所述的荧光探针是标记了两个荧光基团的DNA探针,其中一个标记在探针的5'端,另一个荧光基因和它的相距7 bases或以上,两者可构成能量传递结构,即5'端荧光基因所发出的荧光可被另一荧光基因吸收或抑制。 The fluorescent probe is a labeled DNA probe of the two fluorophores, one of the tags at the 5 'end of the probe, the other fluorescent gene and its 7 bases apart or more, both of which may constitute energy transfer structure , i.e., the 5 'end of the gene emitted fluorescence can be absorbed or suppressed fluorescence of another gene. 探针的3'端羟基(-OH)已被去除或封闭,不具有延伸能力;长度为15~50bases;最好是18~35bases。 Probe 3 'terminal hydroxyl group (-OH) has been removed or blocked, do not have the ability to extend; length of 15 ~ 50bases; preferably 18 ~ 35bases.

所述的缓冲液体系是由pH=5.0~10.0之间的稳定缓冲对,Mg离子和其它促进反应的离子组成,pH值最适选择是PH=7.0~9.0。 The buffer system is a buffer between the stable pH = 5.0 to 10.0 pairs, Mg ions, and other ions facilitate the reaction composition, pH, optimum choice is PH = 7.0 ~ 9.0.

阳性模板系列,是指予先经过数量标定的阳性质控标准品。 Positive template series, means to go through a number of calibration standard positive control. 阳性模板的最好是含有已知数量目的基因的临床标本;阳性模板的另一选择是经过提取、纯化,预先测定好数量的核酸标本;阳性模板的再一选择是经过基因工程克隆、纯化和定量的目的基因。 Template is preferably positive clinical samples containing known quantities of the target gene; another choice is the result of positive template extraction, purification, a good number of nucleic acids sample is measured in advance; then a positive selection template is genetically engineered clones, purified, and quantification of the gene.

阴性模板是指阴性质控制标准品。 Negative template is a negative nature control standards. 阴性模板的较好选择是经过确证为阴性的临床标本;阴性模板的另一选择是经过提取、纯化、并经确证为阴性的核酸标本;阴性模板的再一选择是纯净H2O。 Preferably selected template negative clinical specimens are negative after confirmation; another choice is the result of negative template extraction, purification, and confirmed negative by a nucleic acid specimen; then select a template negative pure H2O.

在反应体系上最好用覆盖剂覆盖。 Preferably covered with a coating agent in the reaction system. 覆盖剂是指用于覆盖于反应体系液面,防止蒸发,与水不相溶的油性覆盖剂。 Cover means for covering agent in the reaction system level, to prevent the evaporation of the oily coating agent, and water-immiscible. 较好选择是固体或液体石蜡。 Selecting a solid or preferably liquid paraffin.

本发明中,PCR反应最好是二步法PCR,包括变性和退火/延伸两步。 In the present invention, PCR reactions are preferably two-step PCR, including denaturation and annealing / extension steps. 变性的温度是85℃~99℃,时间10秒~300秒;退火/延伸的温度是37℃~75℃,时间是10秒~600秒,循环次数是15~60次。 Denaturation temperature is 85 ℃ ~ 99 ℃, time of 10 seconds to 300 seconds; temperature of the annealing / extension was 37 ℃ ~ 75 ℃, time is 10 seconds to 600 seconds, the number of cycles 15 to 60 times. 最好是变性温度89℃~95℃,时间20秒~120秒;退火延伸的温度50℃~70℃,时间30秒~200秒,循环次数30~50次。 Preferably denaturation temperature 89 ℃ ~ 95 ℃, time of 20 to 120 seconds; annealing extension temperature 50 ℃ ~ 70 ℃, time of 30 seconds to 200 seconds, the number of cycles 30 to 50 times.

本发明中,荧光激光光是采用单色光,波长λ=300~700nm,检测波长λ=350~800nm。 In the present invention, the fluorescent monochromatic laser light is employed, the wavelength λ = 300 ~ 700nm, detection wavelength λ = 350 ~ 800nm. 荧光激发光较好的波长λ=450~600nm,检测波长λ=480~650nm。 Fluorescence excitation light wavelength is preferably λ = 450 ~ 600nm, detection wavelength λ 480 ~ 650nm =.

本发明中,可定量的核酸起始拷贝数范围为1~1011拷贝/ml。 In the present invention, the nucleic acid can be quantified starting copy number ranging from 1 to 1011 copies / ml. 可定量的核酸起始拷贝数较好的范围为103~109拷贝/ml。 Quantifiable starting copy number of nucleic acid is preferably in the range of 103 to 109 copies / ml.

本发明还提供了一种用于荧光定量PCR方法的试剂盒,它包括DNA聚合酶,荧光PCR反应液,阳性模板,阴性模板,以及根据待测核酸的特异性序列设计的PCR引物和荧光引物,其中该荧光PCR反应液中镁离子的浓度为15-20mM,Taq酶用量为8-10单位/反应。 The present invention also provides a kit for the quantitative PCR, which comprises a DNA polymerase, a fluorescent PCR reaction solution, the positive template and negative template, and a test nucleic acid sequence according to the specific design of PCR primers and fluorogenic primers wherein the phosphor concentration of the PCR reaction solution of magnesium ions 15-20mM, Taq enzyme concentration of 8-10 units / reaction.

本发明的试剂盒的另一使用方法,是用核酸提取试剂从临床标本中提取核酸(如果是RNA标本,须经过逆转录,转成cDNA),加入一管荧光PCR反应液中,再加入高温聚合酶,覆盖剂等,经离心混合后,放入PCR-荧光检测-电脑分析一体化仪器,开始PCR过程并进行实时荧光检测,测定信号达到检测设定阀值时的循环参数Ct;同时用系列阳性和阴性模板Ct测值为纵坐标,阳性模板的初始核酸量的自然对数值为横坐标,制成标准曲线,即可从标准曲线上查得样品的起始拷贝数。 Another method of using the kit of the present invention, a nucleic acid extraction reagent is a nucleic acid extracted from clinical specimens (specimens if RNA, subject to reverse, turn into the cDNA), was added a fluorescent PCR reaction mixture, temperature was added polymerase, covers and the like, after mixing centrifugation, fluorescence detection into PCR- - computer integrated analysis instruments, and real-time PCR process starts fluorescence detection, the measurement signal reaches a cycle time of detection parameter set threshold Ct; simultaneously with series of positive and negative templates Ct measured value for the vertical, the initial amount of nucleic acid template positive natural logarithm of the abscissa, the standard curve is made, starting copy number can check the sample obtained from the standard curve.

本试剂盒是针对临床疾病进行的特异性设计,是根据已知的疾病特异性基因系列,经过基因库检索、分析和设计,选择了特异、高效的目的基因片段作为扩增、检测对象,并在其中选择了一对PCR引物和一条荧光探针,探针位于一对PCR引物之间。 This kit is designed for a specific clinical disease is based on a known disease-specific gene family, gene bank after retrieval, analysis and design, the selection of specific and efficient amplification as a target gene, target detection, and in which we selected PCR primers and a fluorescent probe pair of probes are located between the pair of PCR primers.

本发明提供了一种用于淋球菌检测的试剂盒,其PCR引物和荧光引物是根据下述特异性序列设计的:1 GCTACGCATA CCCGCGTTGC TTTGCTGTTC TCGACTGGGC AATTTTCCAG51 TGTCAAACCT TTGGTCTTGG TTTCCAACAG GTCTAGGGTG CGCTCTGCTT101 CGGCTCTCTG CTGTTTCAAG TCGTCCAGCT CGTTCTTGAC GCTCCATATC151 GCTATGAACA GCCCTGCTAT GACTATCAAC CCTGCCGCCG ATATACCTAG201 CAAGCTCCAC AGATAGGGCT TGAATACTGC CTTGCTCATG CGTAACTGCC251 GGGCGTTTAT ATCGGCGGTT ATTTTCTGCT CGCTTTGCTT CAATGCCTCG301 TTGATATTTT TCCGTAACGT CTCTAAGTCT GCTTTCGTTT GTTGCTCTAT351 GCTGGCGGCT TCGGTGCGTG ATGTCTGCTC GAAGGTCTTC G其中,引物序列最好为:PNG1:5'GCT ACG CAT ACC CGC GTT GC 3' 20bpsPNG2:5'CGA AGA CCT TCG AGC AGA CA 3' 20bps荧光探针序列最好为:FPNG:5'CAA GTC GTC CAG CTC GTT CTT GAC 3'24bps本发明提供了一种用于乙肝病毒检测的试剂盒,其PCR引物和荧光引物是根据下述特异性序列设计的:1 ATCCTGCTGC TATGCCTCAT CTTCTTGTTG GTTCTTCTGG ACTATCAAGG51 TATGTTGCCC GTTTGTCCTC The present invention provides a kit for detecting Neisseria gonorrhoeae, the PCR primers and fluorescent primers designed according to the following specific sequence: 1 GCTACGCATA CCCGCGTTGC TTTGCTGTTC TCGACTGGGC AATTTTCCAG51 TGTCAAACCT TTGGTCTTGG TTTCCAACAG GTCTAGGGTG CGCTCTGCTT101 CGGCTCTCTG CTGTTTCAAG TCGTCCAGCT CGTTCTTGAC GCTCCATATC151 GCTATGAACA GCCCTGCTAT GACTATCAAC CCTGCCGCCG ATATACCTAG201 CAAGCTCCAC AGATAGGGCT TGAATACTGC CTTGCTCATG CGTAACTGCC251 GGGCGTTTAT ATCGGCGGTT ATTTTCTGCT CGCTTTGCTT CAATGCCTCG301 TTGATATTTT TCCGTAACGT CTCTAAGTCT GCTTTCGTTT GTTGCTCTAT351 GCTGGCGGCT TCGGTGCGTG ATGTCTGCTC GAAGGTCTTC G wherein the primer sequence is preferably: PNG1: 5'GCT ACG CAT ACC CGC GTT GC 3 '20bpsPNG2: 5'CGA AGA CCT TCG AGC AGA CA 3 '20bps fluorescent probe sequence preferably: FPNG: 5'CAA GTC GTC CAG CTC GTT CTT GAC 3'24bps present invention provides a kit for detecting hepatitis B virus, the PCR primers and fluorescent primers were designed according to the following specific sequence: 1 ATCCTGCTGC TATGCCTCAT CTTCTTGTTG GTTCTTCTGG ACTATCAAGG51 TATGTTGCCC GTTTGTCCTC TAATTCCAGG TACTTCAACA ACCAGCACGG101 GACCATGCAG AACCTGCACG ACTCCTGCTC AAGGAACCTC TATGTATCCC151 TCCTGTTGCT GTACCAAACC TTCGGACGGA AATTGCACCT GTATTCCCAT201 CCCATCATCT TGGGCTTTCG GAAAATTCCT ATGGCAGTGG GCCTCAGCCC251 GTTTCTCCTG GCTCAGTTTA CTAGTGCCAT TTGTTCAGTG GTTCGTAGGG301 CTTTCCCCCA CTGT其中,引物序列最好为:PHBV1:5'ATCCTGCTGCTATGCCTCATCTT 3',23bpsPHBV2:5'ACAGTGGGGGAAAGCCCTACGAA 3',23bps荧光探针序列最好为:FPHBV:5'TGGCTAGTTTACTAGTGCCATTTG 3',25bps本发明提供了一种用于丙肝病毒检测的试剂盒,其PCR引物和荧光引物是根据下述特异性序列设计的:1 CTTCACGCAG AAAGCGTCTA GCCATGGCGT TAGTATGAGT GTCGTGCAGC51 CTCCAGGACC CCCCCTCCCG GGAGAGCCAT AGTGGTCTGC GGAACCGGTG101 AGTACACCGG AATTGCCAGG ACGACCGGGT CCTTTCTTGG ATCAACCCGC151 TCAATGCCTG GAGATTTGGG CGTGCCCCCG CGAGACTGCT AGCCGAGTAG201 TGTTGGGTCG CGAAAGGCCT TGTGGTACTG CCTGATAGGG TGCTTGCGAG251 TGCCCCGGGA GGTCTCGTAG A其中,引物序列最好为:PHCV1:5'CTTCACGCAGAAAGCGTCTAGC,22b TAATTCCAGG TACTTCAACA ACCAGCACGG101 GACCATGCAG AACCTGCACG ACTCCTGCTC AAGGAACCTC TATGTATCCC151 TCCTGTTGCT GTACCAAACC TTCGGACGGA AATTGCACCT GTATTCCCAT201 CCCATCATCT TGGGCTTTCG GAAAATTCCT ATGGCAGTGG GCCTCAGCCC251 GTTTCTCCTG GCTCAGTTTA CTAGTGCCAT TTGTTCAGTG GTTCGTAGGG301 CTTTCCCCCA CTGT wherein the primer sequence is preferably: PHBV1: 5'ATCCTGCTGCTATGCCTCATCTT 3 ', 23bpsPHBV2: 5'ACAGTGGGGGAAAGCCCTACGAA 3', 23bps fluorescent probe sequence preferably: FPHBV: 5'TGGCTAGTTTACTAGTGCCATTTG 3 ', 25bps present invention provides a kit for detecting hepatitis C virus, which is a fluorescent PCR primers and primers designed according to the following specific sequence: 1 CTTCACGCAG AAAGCGTCTA GCCATGGCGT TAGTATGAGT GTCGTGCAGC51 CTCCAGGACC CCCCCTCCCG GGAGAGCCAT AGTGGTCTGC GGAACCGGTG101 AGTACACCGG AATTGCCAGG ACGACCGGGT CCTTTCTTGG ATCAACCCGC151 TCAATGCCTG GAGATTTGGG CGTGCCCCCG CGAGACTGCT AGCCGAGTAG201 TGTTGGGTCG CGAAAGGCCT TGTGGTACTG CCTGATAGGG TGCTTGCGAG251 TGCCCCGGGA GGTCTCGTAG A wherein the primer sequence is preferably: PHCV1: 5'CTTCACGCAGAAAGCGTCTAGC, 22b psPHCV2:5'TCTACGAGACCTCCCGGGGCAC,22bps荧光探针序列最好为:FPHCV:5'GAGAGCCATAGTGGTCTGCGGAAC,24bps本发明提供了一种用于沙眼衣原体检测的试剂盒,其PCR引物和荧光引物是根据下述特异性序列设计的:1 CGATGATTTG AGCGTGTGTA GCGCTGAAGA AAATTTGAGC AATTTCATTT51 TCCGCTCGTT TAATGAGTAC AATGAAAATC CATTGCGTAG ATCTCCGTTT101 CTATTGCTTG AGCGTATAAA GGGAAGGCTT GATAGTGCTA TAGCAAAGAC151 TTTTTCTATT CGCAGCGCTA GAGGCCGGTC TATTTATGAT ATATTCTCAC201 AGTCAGAAAT TGGAGTGCTG GCTCGTAT其中,引物序列最好为:PCT1:5'CGA TGA TTT GAG CGT GTG TAG CG 3'23bpsPCT2:5'ATA CGA GCC AGC ACT CCA ATT TC 3'23bps荧光探针序列最好为:FPCT:5'TGA GCA ATT TCA TTT TCC GCT CG 3'23bps本发明提供了一种用于解脲支原体检测的试剂盒,其PCR引物和荧光引物是根据下述特异性序列设计的:1 TTTATAAGGA GATAATGATT ATATGTCAGG ATCATCAAAT CAATTCACTC51 CAGGTAAATT AGTACCAGGA GCAATTAACT TCGCTGAAGG CGAAAATGTG psPHCV2: 5'TCTACGAGACCTCCCGGGGCAC, 22bps fluorescent probe sequence preferably: FPHCV: 5'GAGAGCCATAGTGGTCTGCGGAAC, 24bps present invention provides a kit for the detection of Chlamydia trachomatis which PCR primers and fluorogenic primers are specific sequences according to the following design: 1 CGATGATTTG AGCGTGTGTA GCGCTGAAGA AAATTTGAGC AATTTCATTT51 TCCGCTCGTT TAATGAGTAC AATGAAAATC CATTGCGTAG ATCTCCGTTT101 CTATTGCTTG AGCGTATAAA GGGAAGGCTT GATAGTGCTA TAGCAAAGAC151 TTTTTCTATT CGCAGCGCTA GAGGCCGGTC TATTTATGAT ATATTCTCAC201 AGTCAGAAAT TGGAGTGCTG GCTCGTAT wherein the primer sequence is preferably: PCT1: 5'CGA TGA TTT GAG CGT GTG TAG CG 3'23bpsPCT2 : 5'ATA CGA GCC AGC ACT CCA ATT TC 3'23bps fluorescent probe sequence preferably: FPCT: 5'TGA GCA ATT TCA TTT TCC GCT CG 3'23bps present invention provides a method for the detection of Ureaplasma urealyticum a kit PCR primers and fluorescent primers designed according to the following specific sequence: 1 TTTATAAGGA GATAATGATT ATATGTCAGG ATCATCAAAT CAATTCACTC51 CAGGTAAATT AGTACCAGGA GCAATTAACT TCGCTGAAGG CGAAAATGTG

101 ATGAACGAAG GTAGAGAAGC AAAAGTAATC AGCATTAAAA ATACTGGTGA151 CCGTCCTATC CAAGTTGGAT CACATTTGCA CTTATTTGAA ACAAATAGTG201 CATTAGTATT CTTTGATGAA AAAGGAAACG AAGACAAAGA ACGTAAAGTT251 GCTTATGGAC GTCGTTTCGA TATTCTC其中,引物序列最好为:PUU1:5'TTATAAGGAGATAATGATTATGTG,25bpsPUU2:5'GAGAATATCGAAACGACGTCCAT,24bps荧光探针序列最好为:FPUU:5'GGTAGAGAAGCAAAAGTAATCAGCA,25bps本发明提供了一种用于结核杆菌检测的试剂盒,其PCR引物和荧光引物是根据下述特异性序列设计的:1 TCGCCCGTCT ACTTGGTGTT GCTGCGCGGA GACGGTGCGT AAGTGGGTGC51 GCCAGGCGCA GGTCGATGCC GGCGCACGGC CCGGGACCAC GACCGAAGAA101 TCCGCTGAGA TAAAGCGCTT GCGGCGGGAC AACGCCGAAT TGCGAAGGGC151 GAACGCGATT TTAAAGACCG CGTCGGCTTT CTTCGCGGCC GAGCTCGACC201 GGCCAGCACG CTAATTACCC GGTTCATCGC CGATCATCAG GGCCACCGCG251 AGGGCCCCGA TGGTTTGCGG TGGGGTGTCG AGTCGATCTG CACACAGCTG301 ACCGAGCTGG GTGTGCCGAT CGCCCCATCG ACCTACTACG ACCACATCA其中,引物序列最好为:PTB1:5'TCGCCCGTCTACTTGGTGTT 3'20个 101 ATGAACGAAG GTAGAGAAGC AAAAGTAATC AGCATTAAAA ATACTGGTGA151 CCGTCCTATC CAAGTTGGAT CACATTTGCA CTTATTTGAA ACAAATAGTG201 CATTAGTATT CTTTGATGAA AAAGGAAACG AAGACAAAGA ACGTAAAGTT251 GCTTATGGAC GTCGTTTCGA TATTCTC wherein the primer sequence is preferably: PUU1: 5'TTATAAGGAGATAATGATTATGTG, 25bpsPUU2: 5'GAGAATATCGAAACGACGTCCAT, 24bps fluorescent probe sequence preferably: FPUU: 5'GGTAGAGAAGCAAAAGTAATCAGCA, 25bps present invention provides a kit for detecting Mycobacterium tuberculosis, which is a fluorescent PCR primers and primers designed according to the following specific sequence: 1 TCGCCCGTCT ACTTGGTGTT GCTGCGCGGA GACGGTGCGT AAGTGGGTGC51 GCCAGGCGCA GGTCGATGCC GGCGCACGGC CCGGGACCAC GACCGAAGAA101 TCCGCTGAGA TAAAGCGCTT GCGGCGGGAC AACGCCGAAT TGCGAAGGGC151 GAACGCGATT TTAAAGACCG CGTCGGCTTT CTTCGCGGCC GAGCTCGACC201 GGCCAGCACG CTAATTACCC GGTTCATCGC CGATCATCAG GGCCACCGCG251 AGGGCCCCGA TGGTTTGCGG TGGGGTGTCG AGTCGATCTG CACACAGCTG301 ACCGAGCTGG GTGTGCCGAT CGCCCCATCG ACCTACTACG ACCACATCA wherein the primer sequence is preferably: PTB1: 5'TCGCCCGTCTACTTGGTGTT 3'20 a 碱基PTB2:5'TGATGTGGTCGTAGTAGGTC 3'20个碱基荧光探针序列最好为:FPTB:5'ACA ACG CCG AAT TGC GAA GGG C 3'22bps本发明是使用一套经过优化改进的试剂,组成PCR试剂盒。 Base PTB2: 5'TGATGTGGTCGTAGTAGGTC 3'20 bases fluorescent probe sequence preferably: FPTB: 5'ACA ACG CCG AAT TGC GAA GGG C 3'22bps the present invention is the use of an optimized improved reagent composition PCR kit. 经改进的试剂配方,具有更强的PCR扩增能力和更高的扩增效率。 Improved reagent formulation, with more amplified PCR amplification efficiency and higher capacity. 在较宽的起始模板浓度范围内(拷贝数0-109/毫升)起始模板和产物的量可以保持良好的相关性,有利于实现准确的PCR定量。 In a wide range of the amount of the initial template concentration (copy number 0-109 / ml) starting template and the product can be kept good correlation, is conducive to accurate PCR quantification. 该试剂盒可以对目的核酸序列进行特异性的体外扩增(PCR),放大目的核酸,直至可以被检出。 The kit may be specific to the nucleic acid sequence in vitro amplification (the PCR), amplifying the nucleic acid until it can be detected. 同时又具有可检测的荧光信号,可以用于核酸定量和定性检测;此外,本试剂盒可以使用常规的荧光检测仪,在PCR反应开始前和PCR反应结束之后各测定一次荧光,即可经计算,得出定量或定性的结果;本试剂盒也可以使用自动化的PCR-荧光检测-电脑分析一体化仪器,经过连续实时检测而得到定量结果;再者,本试剂盒系针对临床疾病诊断而设计,具有特异、灵敏、准确、抗污染、快速等特点。 While having a detectable fluorescent signal it can be used for qualitative and quantitative detection of nucleic acids; in addition, the present kit using conventional fluorescence detector, a fluorescence measured after each PCR reaction and the PCR reaction completed before the beginning, can be calculated , obtain quantitative or qualitative result; this kit may be used in automated fluorescence detection PCR- - computer integrated analysis instruments, through continuous real-time quantitative detection result; Furthermore, the present kit system designed for clinical diagnosis with specific, sensitive, accurate, anti-pollution, fast and so on.

使用本试剂盒的有益效果是,可以准确、快速、特异、方便地检测出临床疾病相关的特异性核酸序列。 Using the beneficial effects of the kit may be accurate, rapid, specific and easily detect the specific nucleic acid sequences associated with clinical disease. 准确定量范围为0-109拷贝/毫升,误差<300%。 Accurate quantitation range 0-109 copies / mL, error <300%. 抗污染性强,对仪器设备的要求低,适用于在各级医疗、科研机构推广应用。 Anti-pollution, low demand for equipment is suitable for application in all levels of medical and scientific research institutions.

附图说明 BRIEF DESCRIPTION

:图1是显示荧光PCR的原理的图,R:荧光报告基团;Q:荧光淬灭基团。 : FIG. 1 is a diagram showing the principle of fluorescence PCR, R: fluorescent reporter group; Q: quencher group. 其中:(1)5′→3′外切核酸酶活性用于荧光定量PCR的Taq酶不仅有延伸DNA引物的活性(DNA聚合酶活性),还有5′→3′外切核酸酶活性,可以在链延伸过程中实现链替换,并将被替换的单链切断。 Wherein: (1) 5 '→ 3' exonuclease activity of Taq for quantitative PCR enzyme is active only (DNA polymerase activity) of a DNA primer extension, as well as 5 '→ 3' exonuclease activity, chain may be implemented in alternative chain extension process, and is replaced by a single strand cutting.

(2)荧光标记引物这条探针的5′端和3′端分别标记了一个特殊基团,5′端的基团称为荧光指示基团(R),3′端的基团称为荧光抑制基团(Q)。 (2) fluorescent labeled primer of this probe is 5 'and 3' ends are labeled with a particular group, the 5 'end group are referred fluorescent indicator group (R & lt), the 3' end of the group referred to as fluorescence inhibition group (Q). 当这条探针保持完整时,Q基团将抑制R基团的荧光信号,一旦探针被切断,抑制作用消失,R基团的荧光信号就可以被检测到。 When this probe is intact, Q suppressing fluorescence signal group R groups, once the probe is cut, the inhibition disappeared, the fluorescent signal of the R groups can be detected. (3)荧光PCR反应的实现上述荧光标记探针根据碱基配对原理与扩增产物核酸序列结合。 (3) Fluorescent PCR reactions to achieve the above fluorescently labeled probes bind to the amplification product a nucleic acid sequence according to the principle of base pairing. PCR反应开始后,随着链的延伸,Taq酶沿着DNA模板移动到荧光标记探针结合位置,发挥它的5′→3′外切核酸酶活性,将荧光探针切断,释放出R基团的荧光信号。 After the start of the PCR reaction, along with chain elongation, Taq polymerase moves along the template DNA bound to the fluorescently labeled probe position, exerts its 5 '→ 3' exonuclease activity, the fluorescent probe is cut, releasing group R fluorescent signal groups. 被释放的游离R基团的数目和PCR产物的数量是一对一的关系,因此R基团的荧光信号强弱就与PCR产物数量成正比关系,测量出前者就可以推算出后者。 The number and quantity of the PCR product free of the R groups is one to one relationship is released, the fluorescent signal intensity of the R groups on the relationship proportional to the number of PCR products, the former can be measured calculate the latter. 这就是荧光定量PCR的原理。 This is the principle of quantitative PCR. 图2是实施例1中的荧光PCR定量标准曲线,1g(起始拷贝)。 FIG 2 is a quantitative standard PCR fluorescence curves in Example 1, 1g (initial copy).

试剂盒的配制过程如下:配制5XPCR反应液(50mM Tris-HCl(pH8.0)、100mM MgCl2、250mM KCl、1mg/ml明胶),再配成HBV-PCR反应液:每人份反应液:引物PHBV1,PHBV2(25pmol/μl) 各0.4μl荧光探针FPHBV(20pmol/μl) 0.5μldNTPs(2mM) 5μlTaq酶(10单位) 5μl5×PCR反应缓冲液 10μlddH2O 29.1μl总量 50μl配制DNA裂解液(50mM NaOH、10mM Tris-HCl(pH8.0)、1%Triton X-100、1%NP-40、0.5mM EDTA(pH8.0))阴性质控标准品的制备:取HBV阴性献血员的血清,经乙肝两对半免疫测定,各项指标均为阴性;用PHBV1和PHBV2做HBV PCR检查也为阴性。 The kit of the preparation process is as follows: the reaction solution is formulated 5XPCR (50mM Tris-HCl (pH8.0), 100mM MgCl2,250mM KCl, 1mg / ml gelatin), adding to the HBV-PCR reaction solution: The reaction solution per parts: Primer PHBV1, PHBV2 (25 pmol/μl) of each fluorescent probe 0.4μl FPHBV (20 pmol/μl) 0.5μldNTPs (2mM) 5μlTaq enzyme (10 units) 5μl5 × PCR reaction buffer 50μl 10μlddH2O 29.1μl total DNA prepared lysis buffer (50mM NaOH preparation Tris-HCl (pH8.0), 1% Triton X-100,1% NP-40,0.5mM EDTA (pH8.0)) of negative quality control standards 10mM: take serum HBV negative donors by immunoassay two pairs of semi-hepatitis B, the indicators were negative; do HBV PCR inspection PHBV1 and PHBV2 also negative.

阳性质控标准品的制备:采用HBV病人强阳性血清,经定值血清标准品(100pg/ml)(中国药品生物制品检定所)标定和系列稀释而获得阳性质控标准品。 Preparation of the standard positive control: using strong positive HBV patient serum, the standard value of serum (100pg / ml) (China Pharmaceutical and Biological Products) to obtain dilution series of calibration and positive control standard. 2、使用乙肝病毒荧光定量PCR诊断试剂盒进行检测用无菌注射器抽取受检者静脉血1毫升,注入无菌1.5ml Eppendoff管,于室温静置2小时,转入4℃静置1小时。 2, the use of quantitative PCR of hepatitis B virus diagnostic kit for the detection subject extraction using sterile syringes 1 ml blood, sterile 1.5ml Eppendoff injection tube, allowed to stand at room temperature for 2 hours and transferred to 4 ℃ standing for 1 hour. 8,000rpm离心5分钟,吸取200μl血清(注意勿带入红细胞)转入另一无菌1.5ml Eppendoff管,即为血清标本。 8,000rpm centrifuged for 5 minutes, suction 200μl serum (careful not into erythrocytes) transferred to another sterile tube 1.5ml Eppendoff, i.e. serum samples. 标本和质控标准品的处理取血清标本40μl,加等量DNA提取液打匀。 Standards and controls the processing of specimen serum samples taken 40μl, equal amount of DNA extract Beat. 沸水浴10分钟,转至4℃静置24小时以保证病毒颗粒充分裂解。 A boiling water bath for 10 minutes, transferred to 4 ℃ for 24 hours to ensure adequate lysis of viral particles. 10,000rpm离心5分钟,取上清液2μl做PCR反应。 10,000rpm centrifugation for 5 minutes, the supernatant 2μl doing a PCR reaction. 另取阳性定量质控标准品和阴阳性定量质控标准品各10μl,同上处理。 Another positive quantitative quality control standards and quality control standard quantitative yin and yang of each 10μl, supra process. PCR反应前荧光检测:取Taq酶一管,加入PCR反应液45μl,处理后样品或定量质控标准品2μl,混匀,加30μl石蜡油(如所使用的PCR仪有热盖装置,可不加),离心数秒。 PCR reactions prior to fluorescence detection: Take a Taq enzyme, was added 45 l of the PCR reaction solution, or post-treatment sample 2 l quantitative quality control standards, mix, add 30μl paraffin oil (used as a PCR machine with a thermal cover means may add ), centrifuged for a few seconds. 将该管放入荧光检测仪,读取并记录读数A0。 The tube was placed into the fluorescence detector, read and record the readings A0. 荧光激发波长为487nm,检测波长为525nm。 Fluorescence excitation wavelength of 487nm, the detection wavelength was 525nm. PCR扩增:将各反应管放入PCR仪,按下列条件扩增:93℃→2分钟预变性,然后按93℃45秒→55℃120秒,共做40个循环。 PCR amplification: PCR each reaction tube into the instrument, the following amplification conditions: 93 ℃ → 2 minutes denaturation, and then press 93 ℃ 45 seconds → 55 ℃ 120 seconds and 40 cycles were done. PCR反应后荧光检测检测:待各PCR管充分冷却至室温后,将扩增后的反应管放入荧光检测仪,读取并记录读数A1。 After the PCR, the fluorescence detected by the detector: Each PCR tube be cooled sufficiently to room temperature, the reaction tube into the amplified fluorescence detector, read and record the readings A1. 荧光激发波长为487nm,检测波长为525nm。 Fluorescence excitation wavelength of 487nm, the detection wavelength was 525nm. 计算:AX=A1-A0定性结果判定:样品Ax>N*1.3,判为阳性,否则为阴性。 Calculated: AX = A1-A0 determined qualitative results: Sample Ax> N * 1.3, judged as positive, or negative. 定量计算:以定量阳性质控管的DNA数量(fg)的对数值加1为横坐标(例4fg/μl的对数为lg4=0.602,该管横坐标即为1+lg4=1.602;阴性质控管的横坐标定为0),相应的Ax为纵坐标,用直线联接各定量阳性质控管的数据点,构成标准曲线(图2)。 Quantitative calculation: quantitative amount of DNA in the male nature Controls (FG) plus the value of the abscissa is the number 1 (Example 4fg / μl for lg4 = 0.602, the abscissa is the tube 1 + lg4 = 1.602; Yin properties Controls abscissa as 0), the corresponding vertical axis Ax, each of the data points with a straight line coupling male quantitative nature Controls constituting a standard curve (FIG. 2). 样品的Ax若<N*1.3,则判为阴性;否则以它的Ax值在标准曲线上查出相应横坐标值C,样品的HBV DNA含量(fg/ml)=1000*10(C-1)标本由确诊或怀疑乙肝的病人中采取。 If the sample Ax <N * 1.3, the determination is negative; otherwise isolated by its value Ax standard curve amount of HBV DNA corresponding to the abscissa Found C, the sample (fg / ml) = 1000 * 10 (C-1 ) samples taken from the patients with confirmed or suspected hepatitis B in. 包括急诊病人172例,长期携带者83例,治疗恢复期患者102例,注射过疫苗获免疫者31例,肝癌患者37例,无症状体检者61例和健康献血员21例,其它25例。 Including 172 cases of emergency patients, 83 cases of long-term carriers, treating 102 cases of convalescent patients, vaccinated eligible for immunization were 31 cases, 37 cases of liver cancer patients, 61 cases of asymptomatic volunteers and 21 cases of healthy blood donors, 25 cases of the other. 总病例数达到532例。 The total number of cases to 532 cases.

定量结果采用求算对数平均值的方法来计算平均拷贝数,遇有阴性结果,不参加平均值的统计。 Quantitative results using the method of calculation required to calculate the average number of average copy number in case negative results, do not participate in the statistical average.

乙肝病毒荧光定量PCR检测试剂盒临床应用总结如下:(1)ELISA法共检出HBsAg阳性标本317例,阳性率为59.6%;PCR共检出310例阳性,阳性率为58.3%。 Clinical application of quantitative PCR kit HBV fluorescence summarized as follows: (1) ELISA method were detected HBsAg positive samples 317 cases, the positive rate was 59.6%; 310 cases of positive PCR were detected, the positive rate was 58.3%.

(2)在158例乙肝免疫指标大三阳(HBsAg+/HBeAg+/HBcAb+)的标本中,PCR全部阳性,表明试剂盒用于大三阳诊断的假阴性率为0;在107例乙肝免疫指标全阴性的标本中,PCR全部阴性,表明试剂盒的假阳性率为0。 (2) 158 cases of hepatitis B immune index HBeAg (HBsAg + / HBeAg + / HBcAb +) specimens, all the PCR positive, indicating that the kit false negative diagnosis for HBeAg was 0; 107 cases of hepatitis B immune index Full negative samples, the PCR were all negative, indicating false positive rate kit 0.

(3)在98例乙肝免疫指标小三阳(HBsAg+/HBeAb+/HBcAb+)的标本中,PCR阳性71例,阳性率73%。 (3) 98 cases of hepatitis B immune index three positive (HBsAg + / HBeAb + / HBcAb +) specimens, 71 cases of the PCR positive, the positive rate of 73%. 与国内外报道的肝组织活检阳性率(70%~80%)一致,大大高于普通PCR的30%的阳性率。 Consistent with liver biopsy positive rate (70% to 80%) reported at home and abroad, much higher than the positive rate of 30% of the ordinary PCR. 证明本试剂盒的灵敏度相当高。 The kit proved quite high sensitivity. (4)乙肝免疫指标大三阳(HBsAg+/HBeAg+/HBcAb+)(标志感染期)的标本中,PCR定量的平均拷贝数为1.1×108;(HBsAg+/HBeAg+/HBeAb+/HBcAb+)(标志感染后期)的标本,平均为2.8×107;(HBsAg+/HBeAb+/HBcAb+)(标志长期携带)的标本,平均为8.6×105;(HBsAb+/HBeAb+/HBcAb+)(标志恢复期)的标本,平均为2.3×105。 (4) hepatitis B immune index HBeAg (HBsAg + / HBeAg + / HBcAb +) (flag infection) specimens, PCR quantitative average copy number of 1.1 × 108; (HBsAg + / HBeAg + / HBeAb + / HBcAb +) (flag late stage of infection) specimens, an average of 2.8 × 107; (HBsAg + / HBeAb + / HBcAb +) (in long-run carried) specimens, an average of 8.6 × 105; (HBsAb + / HBeAb + / HBcAb +) (flag recovery) specimens, an average of 2.3 × 105 . 各组之间差异显著,表明定量PCR可以清楚反映乙肝患者的病程变化情况。 Differences between groups significantly, indicating quantitative PCR clearly reflect changes in the course of hepatitis B patients. 进而可以用于临床诊断,治疗方案选择和疗效监控。 In turn can be used for clinical diagnosis, treatment options and efficacy monitoring. 具有很高的临床应用价值。 With high clinical value. 实施例2-人类结核杆菌(TB)荧光PCR检测试剂盒试剂盒的制备:引物序列为:PTB1:5'TCGCCCGTCTACTTGGTGTT 3'20个碱基PTB2:5'TGATGTGGTCGTAGTAGGTC 3'20个碱基荧光探针序列为:FPTB:5'ACA ACG CCG AAT TGC GAA GGG C 3'22bpsPCR扩增区的DNA序列位于TB的IS986基因区。 Example 2 Preparation of human Mycobacterium tuberculosis (TB) fluorescent PCR Kit embodiment the kit: Primer sequences: PTB1: 5'TCGCCCGTCTACTTGGTGTT 3'20 bases PTB2: 5'TGATGTGGTCGTAGTAGGTC 3'20 bases fluorescent probe sequence It is: FPTB: 5'ACA ACG DNA sequence CCG AAT TGC GAA GGG C 3'22bpsPCR amplified region of IS986 located in the gene region TB. 在EMBL的MITS986序列的第176-524位,全长349bps,为多拷贝基因序列。 At position 176-524 of the EMBL sequence MITS986, full length 349bps, multiple copies of the gene sequence. 试剂盒的其它成分如实施例1。 Other components of the kit as described in Example 1.

标本来自临床确诊或拟诊结核病患者的标本,总病例数达到755例,其中127例为非结核对照标本。 Specimens or specimens from clinically suspected diagnosis of TB patients, the total number of cases to 755 cases, 127 cases of non tuberculosis control specimens.

对照诊断标准采用改良罗氏培养法和金胺荧光染液涂片检查法。 Roche Diagnostic criteria using the modified control culture and auramine fluorescent dye smear method.

结核杆菌荧光PCR检测试剂盒临床应用总结如下:荧光PCR法的阳性率为72.3%(454/628),明显高于培养法31.4%(126/401)和涂片法19.9%(125/628),统计学处理有显著差异(P(0.01)。 Mycobacterium tuberculosis Clinical Application of PCR assay reagent cartridge summarized as follows: the positive rate of 72.3% fluorescence PCR methods (454/628), culture was significantly higher than 31.4% (126/401) and smear 19.9% ​​(125/628) there are significant differences (P (0.01), statistically.

在126例培养阳性的标本中,PCR全部阳性,二者符合率为100%;在127例非结核对照标本中,PCR和培养及涂片全部阴性,表明试剂盒假阳性/假阴性率为0。 In the culture-positive specimens of embodiment 126, all the PCR positive, both the rate was 100%; in 127 cases of non-tuberculous control specimens, PCR and all negative culture and smear, the kit showed false positive / false negative rate 0 . 实施例3-淋球菌(NG)荧光PCR检测试剂盒试剂盒的制备:引物序列为:PNG1:5'GCT ACG CAT ACC CGC GTT GC 3'20bpsPNG2:5'CGA AGA CCT TCG AGC AGA CA 3'20bps荧光探针序列为:FPNG:5'CAA GTC GTC CAG CTC GTT CTT GAC 3'24bpsPCR扩增区的DNA序列位于NG的cppB基因区。 Example 3 Preparation of Neisseria gonorrhoeae (NG) fluorescent PCR Kit Kit: Primer sequences: PNG1: 5'GCT ACG CAT ACC CGC GTT GC 3'20bpsPNG2: 5'CGA AGA CCT TCG AGC AGA CA 3'20bps fluorescent probe sequence: FPNG: DNA sequences 5'CAA GTC GTC CAG CTC GTT CTT GAC 3'24bpsPCR amplified region is located in the NG cppB gene region. 在EMBL的序列的第3141-3531位,全长391bps,为多拷贝基因序列。 At EMBL sequence position 3141-3531 of the full-length 391bps, multiple copies of the gene sequence. 试剂盒的其它成分如标本来自临床确诊或拟诊淋病患者的标本,总病例数达到543例,其中20例淋病确诊患者标本。 Other components of the kit specimens from clinically diagnosed as or suspected gonorrhea patient specimens, the total number of cases to 543 cases, 20 cases of gonorrhea diagnosed patient sample.

对照诊断标准为培养法:用血液琼脂培养基培养、分离、鉴定,并做氧化酶试验。 Control culture diagnostic criteria: a blood agar culture medium, isolation, identification, and do the oxidase test.

淋球菌荧光PCR检测试剂盒临床应用总结如下:(1)荧光PCR法的阳性率为65.6%(343/523),明显高于培养法40.7%(213/523)),统计学处理有显著差异(P(0.01)。 Clinical application of Neisseria gonorrhoeae PCR assay kits are summarized as follows: the positive rate (1) of the PCR technique was 65.6% (343/523), culture was significantly higher than 40.7% (213/523)), statistically significant difference (P (0.01).

(2)在20例确诊淋病患者中,荧光PCR全部阳性,二者符合率为100%,而培养法阳性率为70%(14/20),符合率为70%。 (2) In 20 cases of patients diagnosed with gonorrhea, all the PCR positive fluorescence, both was 100%, while culture positive rate was 70% (14/20), the rate was 70%. 实施例4-沙眼衣原体(CT)荧光PCR检测试剂盒试剂盒的制备:引物序列为:PCT1:5'CGA TGA TTT GAG CGT GTG TAG CG 3'23bpsPCT2:5'ATA CGA GCC AGC ACT CCA ATT TC 3'23bps荧光探针序列最好为:FPCT:5'TGA GCA ATT TCA TTT TCC GCT CG 3'23bpsPCR扩增区的DNA序列位于沙眼衣原体质粒pCTT1基因序列(EMBLCTORF)1900-2127位,荧光探针FPCT位于1935-1957位。 Example 4 Preparation embodiment Chlamydia trachomatis (CT) Fluorescence PCR Kit Kit: Primer sequences: PCT1: 5'CGA TGA TTT GAG CGT GTG TAG CG 3'23bpsPCT2: 5'ATA CGA GCC AGC ACT CCA ATT TC 3 '23bps fluorescent probe sequence preferably: FPCT: DNA sequence 5'TGA GCA ATT TCA TTT TCC GCT CG 3'23bpsPCR amplified region of C. trachomatis plasmid located pCTT1 gene sequence (EMBLCTORF) 1900-2127 bit fluorescent probe FPCT located 1935-1957 bit. 全长228bps,为多拷贝基因序列。 Full length 228bps, multiple copies of the gene sequence. 其它如实施例1。 As in Example 1 the other embodiments.

应用中山医科大学达安基因诊断中心研制的沙眼衣原体荧光PCR诊断试剂盒,对132例沙眼衣原体病人的临床标本进行荧光PCR检测,并同时进行沙眼衣原体培养检查,结果显示荧光PCR检测法的阳性率为51.5%(68/132),培养法的阳性率为24.2%(32/132)。 Tat gene Diagnostic Center of Zhongshan Medical University developed applications Chlamydia trachomatis fluorescent PCR diagnostic kits, Chlamydia trachomatis clinical samples for 132 cases of patients PCR assay, and at the same time Chlamydia trachomatis culture examination showed positive rate of fluorescence PCR assay It was 51.5% (68/132), culture positive rate was 24.2% method (32/132).

表:临床标本沙眼衣原体检测结果:例数 阳性例数 阳性率PCR法检测 132 68 51.5%CT培养 132 32 24.2%荧光PCR法与培养结果比较:在132例培养标本中,荧光PCR阳性为68例,阳性率为51.5%,其中32例培养阳性的标本,荧光PCR全部为阳性,二者的符合率为100%。 Table: Clinical samples Chlamydia trachomatis test results: Example Number of positive cases positive rate of PCR, 132 68 51.5% CT culture 13232 24.2% fluorescence PCR method with culture results of comparison: In 132 cases of culture samples, the fluorescent PCR positive in 68 cases the positive rate was 51.5%, of which 32 cases of culture-positive specimens, all PCR fluorescent positive, both was 100%. 在100例CT培养阴性的标本中,荧光PCR法阳性34例。 CT specimens were culture negative in 100 cases, 34 cases of positive fluorescent PCR method.

我们对10份阳性样本和10份阴性样本重复进行PCR检测2次,结果重复性达100%,标明试剂盒的重复性和稳定性较好。 We parts 10 parts 10 positive samples and negative samples PCR analysis was repeated twice, 100% reproducible results, indicating good reproducibility and stability of the kit.

以上结果说明,荧光PCR检测临床标本中沙眼衣原体,阳性检出率明显高于培养法,与临床符合率高,且具有简便快速的优点。 These results suggest that clinical specimens PCR assay Chlamydia trachomatis positive rate significantly higher than the culture, and clinical coincidence rate, and has the advantage of simple and rapid. 实施例5-解脲支原体(UU)荧光PCR检测试剂盒试剂盒的制备:引物序列为:PUU1:5'GCT ACG CAT ACC CGC GTT GC 3'20bpsPUU2:5'CGA AGA CCT TCG AGC AGA CA 3'20bps荧光探针序列为:FPUU:5'CAA GTC GTC CAG CTC GTT CTT GAC 3'24bpsPCR扩增区的DNA序列位于解脲支原体基因序列(EMBL UULOCAB)1-277位,荧光探针FPUU位于135-159位。 Example 5 - Preparation of fluorescent PCR Kit Kit Ureaplasma urealyticum (UU): Primer sequences: PUU1: 5'GCT ACG CAT ACC CGC GTT GC 3'20bpsPUU2: 5'CGA AGA CCT TCG AGC AGA CA 3 ' 20bps fluorescent probe sequence: FPUU: DNA sequences 5'CAA GTC GTC CAG CTC GTT CTT GAC 3'24bpsPCR amplified region is located Ureaplasma urealyticum gene sequence (EMBL UULOCAB) 1-277 bits, a fluorescent probe FPUU located 135- 159. 全长277bps,为多拷贝基因序列。 Full length 277bps, multiple copies of the gene sequence. 其它如实施例1。 As in Example 1 the other embodiments.

应用中山医科大学达安基因诊断中心研制的解脲支原体(UU)荧光PCR诊断试剂盒,对128例我院妇产科门诊患者进行解脲支原体检测,同时用细胞进行培养,结果如下:128例女性生殖道UU检测报告总例数 阳性数 阳性率荧光PCR检测 128 51 39.9%UU培养 128 21 16.4%解脲支原体是女性生殖道常见的病原体。 Application of Ureaplasma urealyticum (UU) Zhongshan Medical University developed Tat gene diagnostic fluorescence PCR diagnostic kit for 128 cases of patients with obstetrics and gynecology outpatients detecting Ureaplasma urealyticum, while cells cultured with the following results: 128 cases female genital tract UU total number of reported cases of positive detection rates of positive PCR assay 128 51 39.9% UU culture 12821 16.4% UU female reproductive tract are common pathogens. 对UU的检测,WHO推荐为细胞培养,但由于对UU的培养需要一定的条件和耗时长,不能满足对性传播疾病的早期快速诊断。 Detection of UU's, WHO recommended cell culture, but the culture of the UU need certain conditions and time-consuming, can not meet the rapid early diagnosis of sexually transmitted diseases. 我们采用中山医科大学达安基因诊断中心研制的解脲支原体荧光PCR诊断试剂盒,对128例高危人群检测结果提示,解脲支原体荧光PCR诊断试剂的特异性和敏感性均高于培养法。 We use the Tat gene Diagnostic Center of Zhongshan Medical University developed UU fluorescent PCR diagnostic kits, 128 patients with high-risk populations suggest a detection result, specificity and sensitivity of PCR diagnostic reagents Ureaplasma urealyticum fluorescence higher than culture. 实施例6-丙型肝炎病毒(HCV)荧光PCR检测试剂盒丙肝病毒荧光PCR诊断试剂盒的组成:选用的引物和荧光探针设计在丙肝病毒基因中的保守区,只对丙肝病毒特异,和其它物种无交叉反应。 Composition (HCV) fluorescence PCR Kit fluorescent PCR of hepatitis C virus diagnostic kit of Example 6 HCV embodiment: the choice of primers and fluorescent probes were designed in the conserved regions of HCV gene, specific for hepatitis C virus only, and no other species cross-reactivity. 荧光探针位于一对引物之间的区域。 Fluorescent probe located in the region between the primer couple. 引物序列为:PHCV1:5'CTTCACGCAGAAAGCGTCTAGC,22bpsPHCV2:5'TCTACGAGACCTCCCGGGGCAC,22bps荧光探针序列最好为:FPHCV:5'GAGAGCCATAGTGGTCTGCGGAAC,24bpsPCR扩增区的DNA序列位于HBV基因5'非翻译区。 Primer sequences: PHCV1: 5'CTTCACGCAGAAAGCGTCTAGC, 22bpsPHCV2: 5'TCTACGAGACCTCCCGGGGCAC, 22bps fluorescent probe sequence preferably: FPHCV: 5'GAGAGCCATAGTGGTCTGCGGAAC, DNA sequence of the amplified region located 24bpsPCR HBV gene 5 'untranslated region. 在EMBL的HCV DNA序列(EMBL S38204)的第62-332位,全长271bps,为单拷贝基因序列。 EMBL sequence of the HCV DNA (EMBL S38204) of 62-332 bits, full length 271bps, single copy gene sequence.

应用中山医科大学达安基因诊断中心研制的HCV荧光PCR诊断试剂盒,对188例我院门诊患者和住院病人进行HCV检测,同时用ELISA检测抗HCV抗体作为对照观察,PCR法阳性率为25%,ELISA法阳性率为22%。 Application of Zhongshan Medical University developed Tat gene diagnosis of HCV PCR for diagnostic kits, for 188 cases of patients outpatients and inpatients HCV testing, while anti-HCV antibodies detected by ELISA as a control was observed, positive PCR method was 25% , ELISA method was 22% positive. 结果如下:荧光PCR 合计+ -ELISA+ 38 3 41ELISA- 9 138 147合计 47 141 188 The results are as follows: Total time PCR + -ELISA + 38 3 41ELISA- 9 138 147 Total 47 141 188

Claims (25)

  1. 1.一种荧光定量聚合酶链式反应方法,包括以下步骤:a、根据待检测靶基因的特异性核酸序列合成一对引物;b、根据待检测靶基因的特异性核酸序列中位于两个引物之间的序列设计、合成一个荧光探针;c、使用步骤a中的引物和步骤b中的荧光探针及其它聚合酶链式反应成份组成荧光聚合酶链式反应体系,其中镁离子的浓度为15-20mM,Taq酶用量为8-10单位/反应;d、从待测标本中提取DNA,或是提取RNA然后逆转录成cDNA,加入步骤c中的反应体系中,经离心混合后测定反应前荧光值;e、将反应管放入聚合酶链式反应仪进行20~50次循环的聚合酶链式反应;反应结束后再测定一次反应管中的荧光值;前后两次测值的差值即代表了本管反应的荧光增值;f、同时用系列阳性和阴性模板也进行步骤d的核酸提取和聚合酶链式反应,测得各自相应的荧光增值,将荧光增 Chain reaction quantitative polymerase 1. A method, comprising the steps of: a, using a pair of primers specific nucleic acid sequence to be detected in accordance with the target gene; B, located between the two specific nucleic acid sequence to be detected in accordance with the target gene design of the sequence between the primers was synthesized a fluorescent probe; C, and the primers used in step b step a fluorescent probe in a polymerase chain reaction and other polymerase chain reaction component composition of the phosphor system, wherein the magnesium ions at a concentration of 15-20mM, Taq enzyme concentration of 8-10 units / reaction; D, extracting DNA from the test sample, or the cDNA reverse transcribed into RNA was extracted and then, in step c was added in the reaction system by centrifugation after mixing measured fluorescence value before the reaction; e, the reaction tube was placed in a polymerase chain reaction instrument for 20 to 50 cycles of polymerase chain reaction; measuring the fluorescence values ​​after completion of the reaction time the reaction tube; the two measured values ​​before and after which represents the difference between the present value of fluorescence of the reaction tube; F, while a series of positive and negative templates also step d nucleic acid extraction and polymerase chain reaction, a respective measured value of the fluorescence, the fluorescence increase 相对于阳性模板的初始核酸量的自然对数值作图,制成标准曲线;g、用上述e步骤中获得的各样品的荧光增值,可在标准曲线上查出相应的样品中的核酸起始拷贝数。 Relative to the initial amount of nucleic acid template positive natural logarithm was plotted to prepare a calibration curve; G, the fluorescence value of each sample obtained in the above step e, the starting nucleic acid may be isolated in the respective sample on the standard curve copy number.
  2. 2.按照权利要求1所述的方法,其特征在于,步骤a中所述的两个引物之间相距50~500个碱基。 2. The method according to claim 1, characterized in that, apart from 50 to 500 bases between two primers in said step a.
  3. 3.按照权利要求1所述的方法,其特征在于,步骤e中所述的聚合酶链式反应进行30~50个循环。 3. The method according to claim 1, wherein, in step e the polymerase chain reaction for 30 to 50 cycles.
  4. 4.按照权利要求1所述的方法,其特征在于,步骤e中所述的聚合酶链式反应进行40个循环。 4. The method according to claim 1, wherein, in step e the polymerase chain reaction was performed for 40 cycles.
  5. 5.按照权利要求1所述的方法,其特征在于,步骤a中所述的引物的长度是16~25个碱基。 5. The method according to claim 1, characterized in that the length of the primer in the step a is 16 to 25 nucleotides.
  6. 6.按照权利要求1所述的方法,其特征在于,步骤a中所述的荧光探针的长度是18~35个碱基。 6. The method according to claim 1, characterized in that the length of the fluorescent probe according to step a 18 to 35 bases.
  7. 7.一种用于荧光定量聚合酶链式反应方法的试剂盒,它包括DNA聚合酶,荧光聚合酶链式反应液,阳性模板,阴性模板,以及根据待测核酸的特异性核酸序列设计的聚合酶链式反应引物和荧光引物,其特征在于,该荧光聚合酶链式反应液中镁离子的终浓度为15-20mM,Taq酶用量为8-10单位/反应。 A kit for quantitative polymerase chain reaction, which comprises a DNA polymerase, the fluorescent polymerase chain reaction solution, the positive template and negative template, the test nucleic acid and designed according to the specific nucleic acid sequence polymerase chain reaction primers and fluorogenic primers, wherein the fluorescent polymerase chain reaction mixture to a final concentration of 15-20mM magnesium ions, Taq polymerase in an amount of 8-10 units / reaction.
  8. 8.按照权利要求7所述的试剂盒,其特征在于,所述的待测核酸为淋球菌DNA,所述的特异性核酸序列为:1 GCTACGCATA CCCGCGTTGC TTTGCTGTTC TCGACTGGGC AATTTTCCAG51 TGTCAAACCT TTGGTCTTGG TTTCCAACAG GTCTAGGGTG CGCTCTGCTT101 CGGCTCTCTG CTGTTTCAAG TCGTCCAGCT CGTTCTTGAC GCTCCATATC151 GCTATGAACA GCCCTGCTAT GACTATCAAC CCTGCCGCCG ATATACCTAG201 CAAGCTCCAC AGATAGGGCT TGAATACTGC CTTGCTCATG CGTAACTGCC251 GGGCGTTTAT ATCGGCGGTT ATTTTCTGCT CGCTTTGCTT CAATGCCTCG301 TTGATATTTT TCCGTAACGT CTCTAAGTCT GCTTTCGTTT GTTGCTCTAT351 GCTGGCGGCT TCGGTGCGTG ATGTCTGCTC GAAGGTCTTC G 8. The kit of claim 7, wherein said test nucleic acid to the DNA of Neisseria gonorrhoeae, said specific nucleic acid sequence is: 1 GCTACGCATA CCCGCGTTGC TTTGCTGTTC TCGACTGGGC AATTTTCCAG51 TGTCAAACCT TTGGTCTTGG TTTCCAACAG GTCTAGGGTG CGCTCTGCTT101 CGGCTCTCTG CTGTTTCAAG TCGTCCAGCT CGTTCTTGAC GCTCCATATC151 GCTATGAACA GCCCTGCTAT GACTATCAAC CCTGCCGCCG ATATACCTAG201 CAAGCTCCAC AGATAGGGCT TGAATACTGC CTTGCTCATG CGTAACTGCC251 GGGCGTTTAT ATCGGCGGTT ATTTTCTGCT CGCTTTGCTT CAATGCCTCG301 TTGATATTTT TCCGTAACGT CTCTAAGTCT GCTTTCGTTT GTTGCTCTAT351 GCTGGCGGCT TCGGTGCGTG ATGTCTGCTC GAAGGTCTTC G
  9. 9.按照权利要求8所述的试剂盒,其特征在于,所述的引物序列为:引物1:5′GCT ACG CAT ACC CGC GTT GC 3′引物2:5′CGA AGA CCT TCG AGC AGA CA 3′ 9. The kit of claim 8, wherein the primer sequences: Primer 1: 5'GCT ACG CAT ACC CGC GTT GC 3 'Primer 2: 5'CGA AGA CCT TCG AGC AGA CA 3 '
  10. 10.按照权利要求8所述的试剂盒,其特征在于,所述的荧光探针的序列为:5′CAA GTC GTC CAG CTC GTT CTT GAC 3′。 10. The kit of claim 8, wherein said fluorescent probe sequence is: 5'CAA GTC GTC CAG CTC GTT CTT GAC 3 '.
  11. 11.按照权利要求7所述的试剂盒,其特征在于,所述的待测核酸为乙肝病毒DNA,所述的特异性核酸序列为:1 ATCCTGCTGC TATGCCTCAT CTTCTTGTTG GTTCTTCTGG ACTATCAAGG51 TATGTTGCCC GTTTGTCCTC TAATTCCAGG TACTTCAACA ACCAGCACGG101 GACCATGCAG AACCTGCACG ACTCCTGCTC AAGGAACCTC TATGTATCCC151 TCCTGTTGCT GTACCAAACC TTCGGACGGA AATTGCACCT GTATTCCCAT201 CCCATCATCT TGGGCTTTCG GAAAATTCCT ATGGCAGTGG GCCTCAGCCC251 GTTTCTCCTG GCTCAGTTTA CTAGTGCCAT TTGTTCAGTG GTTCGTAGGG301 CTTTCCCCCA CTGT 11. The kit of claim 7, wherein said test nucleic acid is the hepatitis B viral DNA, the specific nucleic acid sequence is: 1 ATCCTGCTGC TATGCCTCAT CTTCTTGTTG GTTCTTCTGG ACTATCAAGG51 TATGTTGCCC GTTTGTCCTC TAATTCCAGG TACTTCAACA ACCAGCACGG101 GACCATGCAG AACCTGCACG ACTCCTGCTC AAGGAACCTC TATGTATCCC151 TCCTGTTGCT GTACCAAACC TTCGGACGGA AATTGCACCT GTATTCCCAT201 CCCATCATCT TGGGCTTTCG GAAAATTCCT ATGGCAGTGG GCCTCAGCCC251 GTTTCTCCTG GCTCAGTTTA CTAGTGCCAT TTGTTCAGTG GTTCGTAGGG301 CTTTCCCCCA CTGT
  12. 12.按照权利要求11所述的试剂盒,其特征在于,所述的引物序列为:引物1:5′ATCCTGCTGCTATGCCTCATCTT 3′,引物2:5′ACAGTGGGGGAAAGCCCTACGAA 3′。 12. The kit of claim 11, wherein the primer sequences: Primer 1: 5'ATCCTGCTGCTATGCCTCATCTT 3 ', primer 2: 5'ACAGTGGGGGAAAGCCCTACGAA 3'.
  13. 13.按照权利要求11所述的试剂盒,其特征在于,所述的荧光探针的序列为:5′TGGCTAGTTTACTAGTGCCATTTG 3′。 13. The kit of claim 11, wherein said fluorescent probe sequence is: 5'TGGCTAGTTTACTAGTGCCATTTG 3 '.
  14. 14.按照权利要求7所述的试剂盒,其特征在于,所述的待测核酸为丙肝病毒DNA,所述的特异性核酸序列为:1 CTTCACGCAG AAAGCGTCTA GCCATGGCGT TAGTATGAGT GTCGTGCAGC51 CTCCAGGACC CCCCCTCCCG GGAGAGCCAT AGTGGTCTGC GGAACCGGTG101 AGTACACCGG AATTGCCAGG ACGACCGGGT CCTTTCTTGG ATCAACCCGC151 TCAATGCCTG GAGATTTGGG CGTGCCCCCG CGAGACTGCT AGCCGAGTAG201 TGTTGGGTCG CGAAAGGCCT TGTGGTACTG CCTGATAGGG TGCTTGCGAG251 TGCCCCGGGA GGTCTCGTAG A 14. The kit of claim 7, wherein said test nucleic acid is hepatitis C viral DNA, the specific nucleic acid sequence is: 1 CTTCACGCAG AAAGCGTCTA GCCATGGCGT TAGTATGAGT GTCGTGCAGC51 CTCCAGGACC CCCCCTCCCG GGAGAGCCAT AGTGGTCTGC GGAACCGGTG101 AGTACACCGG AATTGCCAGG ACGACCGGGT CCTTTCTTGG ATCAACCCGC151 TCAATGCCTG GAGATTTGGG CGTGCCCCCG CGAGACTGCT AGCCGAGTAG201 TGTTGGGTCG CGAAAGGCCT TGTGGTACTG CCTGATAGGG TGCTTGCGAG251 TGCCCCGGGA GGTCTCGTAG A
  15. 15.按照权利要求14所述的试剂盒,其特征在于,所述的引物序列为:引物1:5'CTTCACGCAGAAAGCGTCTAGC,引物2:5'TCTACGAGACCTCCCGGGGCAC。 15. The kit of claim 14, wherein the primer sequences: Primer 1: 5'CTTCACGCAGAAAGCGTCTAGC, primer 2: 5'TCTACGAGACCTCCCGGGGCAC.
  16. 16.按照权利要求14所述的试剂盒,其特征在于,所述的荧光探针的序列为:5'GAGAGCCATAGTGGTCTGCGGAAC。 16. The kit of claim 14, wherein said fluorescent probe sequence is: 5'GAGAGCCATAGTGGTCTGCGGAAC.
  17. 17.按照权利要求7所述的试剂盒,其特征在于,所述的待测核酸为沙眼衣原体DNA,所述的特异性核酸序列为:1 CGATGATTTG AGCGTGTGTA GCGCTGAAGA AAATTTGAGC AATTTCATTT51 TCCGCTCGTT TAATGAGTAC AATGAAAATC CATTGCGTAG ATCTCCGTTT101 CTATTGCTTG AGCGTATAAA GGGAAGGCTT GATAGTGCTA TAGCAAAGAC151 TTTTTCTATT CGCAGCGCTA GAGGCCGGTC TATTTATGAT ATATTCTCAC201 AGTCAGAAAT TGGAGTGCTG GCTCGTAT 17. The kit of claim 7, wherein said nucleic acid is Chlamydia trachomatis tested the DNA, the specific nucleic acid sequence is: 1 CGATGATTTG AGCGTGTGTA GCGCTGAAGA AAATTTGAGC AATTTCATTT51 TCCGCTCGTT TAATGAGTAC AATGAAAATC CATTGCGTAG ATCTCCGTTT101 CTATTGCTTG AGCGTATAAA GGGAAGGCTT GATAGTGCTA TAGCAAAGAC151 TTTTTCTATT CGCAGCGCTA GAGGCCGGTC TATTTATGAT ATATTCTCAC201 AGTCAGAAAT TGGAGTGCTG GCTCGTAT
  18. 18.按照权利要求17所述的试剂盒,其特征在于,所述的引物序列为:引物1:5′CGA TGA TTT GAG CGT GTG TAG CG 3′,引物2:5′ATA CGA GCC AGC ACT CCA ATT TC 3′ 18. The kit of claim 17, wherein the primer sequences: Primer 1: 5'CGA TGA TTT GAG CGT GTG TAG CG 3 ', primer 2: 5'ATA CGA GCC AGC ACT CCA ATT TC 3 '
  19. 19.按照权利要求17所述的试剂盒,其特征在于,所述的荧光探针的序列为:5'TGA GCA ATT TCA TTT TCC GCT CG 3'。 19. The kit of claim 17, wherein said fluorescent probe sequence is: 5'TGA GCA ATT TCA TTT TCC GCT CG 3 '.
  20. 20.按照权利要求7所述的试剂盒,其特征在于,所述的待测核酸为解脲支原体DNA,所述的特异性核酸序列为:1 TTTATAAGGA GATAATGATT ATATGTCAGG ATCATCAAAT CAATTCACTC51 CAGGTAAATT AGTACCAGGA GCAATTAACT TCGCTGAAGG CGAAAATGTG101 ATGAACGAAG GTAGAGAAGC AAAAGTAATC AGCATTAAAA ATACTGGTGA151 CCGTCCTATC CAAGTTGGAT CACATTTGCA CTTATTTGAA ACAAATAGTG201 CATTAGTATT CTTTGATGAA AAAGGAAACG AAGACAAAGA ACGTAAAGTT251 GCTTATGGAC GTCGTTTCGA TATTCTC。 20. The kit of claim 7, wherein said test nucleic acid to the DNA UU, the specific nucleic acid sequence is: 1 TTTATAAGGA GATAATGATT ATATGTCAGG ATCATCAAAT CAATTCACTC51 CAGGTAAATT AGTACCAGGA GCAATTAACT TCGCTGAAGG CGAAAATGTG101 ATGAACGAAG GTAGAGAAGC AAAAGTAATC AGCATTAAAA ATACTGGTGA151 CCGTCCTATC CAAGTTGGAT CACATTTGCA CTTATTTGAA ACAAATAGTG201 CATTAGTATT CTTTGATGAA AAAGGAAACG AAGACAAAGA ACGTAAAGTT251 GCTTATGGAC GTCGTTTCGA TATTCTC.
  21. 21.按照权利要求20所述的试剂盒,其特征在于,所述的引物序列为:引物1:5'TTATAAGGAGATAATGATTATGTG,引物2:5'GAGAATATCGAAACGACGTCCAT。 21. The kit of claim 20, wherein the primer sequences: Primer 1: 5'TTATAAGGAGATAATGATTATGTG, primer 2: 5'GAGAATATCGAAACGACGTCCAT.
  22. 22.按照权利要求20所述的试剂盒,其特征在于,所述的荧光探针的序列为:5'GGTAGAGAAGCAAAAGTAATCAGCA。 22. The kit of claim 20, wherein said fluorescent probe sequence is: 5'GGTAGAGAAGCAAAAGTAATCAGCA.
  23. 23.按照权利要求7所述的试剂盒,其特征在于,所述的待测核酸为结核杆菌DNA,所述的特异性核酸序列为:1 TCGCCCGTCT ACTTGGTGTT GCTGCGCGGA GACGGTGCGT AAGTGGGTGC51 GCCAGGCGCA GGTCGATGCC GGCGCACGGC CCGGGACCAC GACCGAAGAA101 TCCGCTGAGA TAAAGCGCTT GCGGCGGGAC AACGCCGAAT TGCGAAGGGC151 GAACGCGATT TTAAAGACCG CGTCGGCTTT CTTCGCGGCC GAGCTCGACC201 GGCCAGCACG CTAATTACCC GGTTCATCGC CGATCATCAG GGCCACCGCG251 AGGGCCCCGA TGGTTTGCGG TGGGGTGTCG AGTCGATCTG CACACAGCTG301 ACCGAGCTGG GTGTGCCGAT CGCCCCATCG ACCTACTACG ACCACATCA 23. The kit of claim 7, wherein said test nucleic acid to the DNA of Mycobacterium tuberculosis, the specific nucleic acid sequence is: 1 TCGCCCGTCT ACTTGGTGTT GCTGCGCGGA GACGGTGCGT AAGTGGGTGC51 GCCAGGCGCA GGTCGATGCC GGCGCACGGC CCGGGACCAC GACCGAAGAA101 TCCGCTGAGA TAAAGCGCTT GCGGCGGGAC AACGCCGAAT TGCGAAGGGC151 GAACGCGATT TTAAAGACCG CGTCGGCTTT CTTCGCGGCC GAGCTCGACC201 GGCCAGCACG CTAATTACCC GGTTCATCGC CGATCATCAG GGCCACCGCG251 AGGGCCCCGA TGGTTTGCGG TGGGGTGTCG AGTCGATCTG CACACAGCTG301 ACCGAGCTGG GTGTGCCGAT CGCCCCATCG ACCTACTACG ACCACATCA
  24. 24.按照权利要求23所述的试剂盒,其特征在于,所述的引物序列为:引物1:5′TCGCCCGTCTACTTGGTGTT 3′,引物2:5′TGATGTGGTCGTAGTAGGTC 3′ 24. The kit of claim 23, wherein the primer sequences: Primer 1: 5'TCGCCCGTCTACTTGGTGTT 3 ', primer 2: 5'TGATGTGGTCGTAGTAGGTC 3'
  25. 25.按照权利要求23所述的试剂盒,其特征在于,所述的荧光探针的序列为:5′ACA ACG CCG AAT TGC GAA GGG C 3′ 25. The kit of claim 23, wherein said fluorescent probe sequence is: 5'ACA ACG CCG AAT TGC GAA GGG C 3 '
CN 99100669 1999-02-12 1999-02-12 A fluorescent quantitative polymerase chain reaction method and kit CN1094154C (en)

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EP1236804A1 (en) 2001-03-02 2002-09-04 Boehringer Mannheim Gmbh A method for determination of a nucleic acid using a control
CN1316036C (en) * 2003-01-06 2007-05-16 徐定邦 Gene quantitation method by using PCR as basis
CN100494987C (en) 2003-02-27 2009-06-03 东南大学 immobilized nucleic acid probe for non-labeling detection
CN1661359B (en) 2004-02-25 2013-06-12 陕西西大北美基因股份有限公司 Method for measuring isothermal amplification of nucleic acid quantificationally
CN100458419C (en) 2005-04-26 2009-02-04 浙江大学;杭州博康生物科技有限公司 Mitochondria DNA11778 point mutation detecting reagent case by molecular probe technology
CN100427927C (en) 2005-12-08 2008-10-22 中国农业科学院作物科学研究所 Green smut bug real-time fluorescent quantitative PCR test kit and its use
CN100453653C (en) 2006-05-19 2009-01-21 上海申友生物技术有限责任公司 Gene sequencing method combined with fluorescent quantitative PCR
US8190371B2 (en) 2007-09-07 2012-05-29 Third Wave Technologies, Inc. Methods and applications for target quantification
CN103571938B (en) * 2013-02-25 2016-04-20 哈尔滨医科大学 The method of reverse transcription pcr detection from clinical specimens infected with TB

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