CN102191327A - Kit for forecasting death rate of patients with sepsis and application thereof - Google Patents

Kit for forecasting death rate of patients with sepsis and application thereof Download PDF

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CN102191327A
CN102191327A CN 201110096219 CN201110096219A CN102191327A CN 102191327 A CN102191327 A CN 102191327A CN 201110096219 CN201110096219 CN 201110096219 CN 201110096219 A CN201110096219 A CN 201110096219A CN 102191327 A CN102191327 A CN 102191327A
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王慧娟
解立新
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中国人民解放军总医院
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Abstract

The invention provides a kit for forecasting the death rate of patients with sepsis. The kit comprises a reverse transcription system and an amplification system, wherein the reverse transcription system consists of a reverse transcriptase, a reverse transcription system buffer solution, a ribonucleic acid (RNA) enzyme inhibitor, an hsa-miR-16 stemandloop structure primer and a postdigestive RNA extracting solution; and the amplification system consists of a Taq enzyme-containing miRNA expression quantitative detection mixed solution, an hsa-miR-16 amplification primer, an hsa-miR-16 fluorescently tagged probe, a U6snRNA amplification primer and a U6snRNA fluorescently tagged probe. The invention also provides a method for forecasting the death rate of the patients with sepsis by using the kit. By the method, the quantitative detection is accurate, simple, quick, economic and practical and is convenient to perform. The kit can be further combined with other molecular targets and scoring systems, and then a comprehensive molecular marker kit can be provided to forecast the death rate of the patients with sepsis and is convenient to apply clinically.

Description

一种用于预测脓毒症患者病死率的试剂盒及其用途 A method for predicting mortality in patients with sepsis kits and uses thereof

技术领域 FIELD

[0001] 本发明涉及一种用于预测脓毒症患者病死率的试剂盒及其用途,属于生物医学诊断领域。 [0001] The present invention relates to a kit and its use for predicting mortality in septic patients, belonging to the field of biological medical diagnosis.

背景技术 Background technique

[0002] 脓毒症(Sepsis)是严重创(烧、战)伤、休克、感染、外科大手术患者常见的并发症,进一步发展可导致脓毒性休克、多器官功能障碍综合征(MODS),是多发伤最主要死亡原因之一。 [0002] sepsis (Sepsis) is a serious record (burn, war) trauma, shock, infection, a common complication of major surgery patients, further development may lead to septic shock, multiple organ dysfunction syndrome (MODS), It is one of the major multiple trauma causes of death. 目前,脓毒症已成为临床医学面临的突出难题,全世界每年大约1000人中就有3人发生脓毒症和感染性休克,同时这一数字还以每年1. 5%〜8. 0%的速度上升。 Currently, clinical sepsis has become a prominent problem facing the world about 1,000 people every year there are three people sepsis and septic shock, while this figure to 1.5% ~ 8 per year. 0% the rate rise. 近年来抗感染治疗和器官功能支持技术取得了长足的进步,但重症患者病死率仍高达30%〜70%。 In recent years, anti-infective therapy and organ support technology has made considerable progress, but severe cases mortality is still as high as 30% ~ 70%. 我们前期的研究发现重症脓毒症病死率为31%,而一旦出现休克,病死率则高达75%。 Our previous studies found that severe sepsis mortality rate was 31%, and once shock occurs, the mortality is as high as 75%. 在保健医学领域因保健对象普遍年龄较大,多合并有各种慢性疾病,机体贮备能力下降,一旦感染则很容易继发多器官功能衰竭而危及生命。 Due to the large universal healthcare recipients age, more complicated with a variety of chronic diseases, the body's reserve capacity decline in the health field of medicine, once the infection is very easy secondary to multiple organ failure and life-threatening. 国内的一组保健对象资料回顾性分析发现感染致脓毒症(73. 1%,主要是下呼吸道感染)是导致保健对象继发多器官功能不全的最主要因素,而我们的研究发现一旦合并四个脏器以上衰竭,老年患者的病死率高达80%以上, 而且我们研究证实现有的感染监测指标C-反应蛋白(CRP)和降钙素原(PCT)在动态评价脓毒症病情严重程度方面结果令人沮丧(S印sis/revere Sepsis/Septic Shock =CRP :p = 0. 711,PCT :p = 0. 075)。 A set of domestic health data objects retrospective analysis found that infection-induced sepsis (73.1%, mainly lower respiratory tract infection) is the most important factor leading to a secondary target health of multiple organ dysfunction, and our study found that once the merger more than four organ failure and mortality in elderly patients with up to 80%, and our study confirms the existing monitoring indicators of infection C- reactive protein (CRP) and procalcitonin (PCT) in severe sepsis dynamic evaluation of the condition results extent frustrating aspect (S printed sis / revere Sepsis / Septic Shock = CRP: p = 0. 711, PCT: p = 0. 075). 近年来研究表明,微小RNA (microRNA, miRNA)作为一类新型调节因子在肿瘤的发生发展中起重要作用。 Recent studies show that small RNA (microRNA, miRNA) as a new class of regulatory factors play an important role in the development of tumors. 应用基因芯片或是实时定量聚合酶链式反应(PCR) 技术对miRNA表达谱研究发现,miRNA可助于多种疾病的诊断和预后评估。 CDNA microarray or quantitative real-time polymerase chain reaction (PCR) technique miRNA profile found, miRNA may assist diagnosis and prognosis of expression of a variety of diseases. 脓毒症患者病情发展迅速,病情凶险,对脓毒症患者的早期病情评估,并采取适当的干预治疗将十分有利用改善其预后生存。 Rapid progression of the disease in patients with sepsis, dangerous disease, early to assess the condition of patients with sepsis, and appropriate intervention will be very use to improve the prognosis of survival.

发明内容 SUMMARY

[0003] 本发明的一个目的是提供一种用于早期预测脓毒症患者的病死率的试剂盒。 [0003] An object of the present invention is to provide an early prediction of mortality in patients with sepsis kit for.

[0004] 本发明的另一个目的是提供一种将上述试剂盒用于预测脓毒症患者的观天病死率的方法。 [0004] Another object of the present invention is to provide a kit for predicting the above-described concept day mortality in patients with sepsis method.

[0005] 为达到上述目的,本发明的技术方案是提供一种用于预测脓毒症患者病死率的试剂盒,该试剂盒由反转录系统和扩增系统组成。 [0005] To achieve the above object, the technical solution of the present invention is to provide a method for prediction of mortality in patients with sepsis kits, the kit and amplified by the Reverse Transcription System system. 其中,反转录系统由反转录酶、反转录体系缓冲液、RNA酶抑制剂、dNTP mix,hsa-miR-16茎环结构弓|物和消化后的RNA提取液组成,扩增系统由含Taq酶的miRNA表达定量检测混合液、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液组成。 Wherein the Reverse Transcription System by the reverse transcriptase, reverse transcriptase buffer system, RNA inhibitor, dNTP mix, hsa-miR-16 stem-loop structure bow | RNA extraction solution after the composition of matter and digested amplification system Taq polymerase containing the miRNA expression quantitative detection mixture, hsa-miR-16 and the amplification primer hsa-miR-16 a mixture of fluorescently labeled probes and amplification primers TOsnRNA TOsnRNA fluorescently labeled probe mixture solution.

[0006] 所述反转录系统包括:反转录酶0.5 μ 1、反转录缓冲液0.75 μ 1、RNA酶抑制剂0. 095 μ 1,hsa-miR-16 莲环结构引物1. 5 μ 1、去离子水2. 08 μ l.dNTP mix 0. 075 μ 1、及消化后RNA样品2. 5 μ 1,共计7. 5 μ 1。 [0006] The reverse transcription system comprising: a reverse transcriptase, 0.5 μ 1, reverse transcription buffer, 0.75 μ 1, RNA inhibitors 0. 095 μ 1, hsa-miR-16 primers ring structure lotus 1.5 μ 1, deionized water 2. 08 μ l.dNTP mix 0. 075 μ 1, and after digestion of the RNA sample 2. 5 μ 1, a total of 7. 5 μ 1.

[0007] 所述扩增系统包括:含Taq酶的miRNA表达定量检测混合液IOul、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液Iul和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液Iul。 [0007] The amplification system comprising: containing Taq enzyme mixture quantitative detection of miRNA expression IOul, hsa-miR-16 and the amplification primer hsa-miR-16 mixture Iul fluorescently labeled probes and amplification primers TOsnRNA TOsnRNA fluorescently labeled probe mixture Iul.

[0008] 本发明还提供了一种利用上述试剂盒预测脓毒症患者病死率的的方法,包括以下步骤: [0008] The present invention further provides a method of predicting mortality in septic patients using the above kit, comprising the steps of:

[0009] (1)病例血清样本处理及RNA提取 [0009] (1) Case of serum samples RNA extraction and

[0010] 以促凝管采集促凝血,临床常规血清标本以带分离胶的菲可替管采集,抽取入住I⑶脓毒症患者M小时内的血样以及正常人血样,在4°C下以3000rpm离心15min,将上层血清转移至洁净Ep管,再在4°C下以15,OOOrpm离心30min以去除细胞碎片,吸取上层血清于洁净离心管,-80°C冻存备用;按照mirVana PARIS试剂盒使用说明书从血清中提取总RNA,使用kandrop测定RNA提取液的A260吸光度值和A260/A280的比值(1. 9-2. 1),以评估其浓度;变性琼脂糖凝胶电泳检测RNA的完整性,然后参照DNase I使用说明书,于37°C 对RNA提取液消化30min,于_80°C保存备用; [0010] In procoagulant coagulant collection tube, Ficoll to routine clinical serum samples with gel separator for collection tube, blood samples were drawn and stay within the normal blood I⑶ M sepsis patients hours at 4 ° C for at 3000rpm centrifugal 15min, the upper layer of serum was transferred to a clean tube Ep, then at 4 ° C for at 15, OOOrpm 30min to remove cell debris by centrifugation, the serum to draw the upper clean centrifuge tube, -80 ° C frozen for later use; mirVana PARIS kit according to total RNA was extracted using instructions from the serum using a RNA extraction was measured kandrop A260 absorbance values ​​and the ratio of A260 / A280 of (1. 9-21.), to assess the concentration; denaturing agarose gel electrophoresis of intact RNA resistance, and instructions for use referring to DNase I at 37 ° C to extract RNA digestion 30min, were stored at _80 ° C;

[0011 ] (2) Hsa-miR-16cDNA 的合成 [0011] Synthesis of (2) Hsa-miR-16cDNA of

[0012] 反转录体系包括:反转录酶0.5μ 1、反转录缓冲液0.75μ 1、RNA酶抑制剂0. 095 μ 1,hsa-miR-16 莲环结构引物1. 5 μ 1、去离子水2. 08 μ l.dNTP mix 0. 075 μ 1、及消化后RNA样品2. 5 μ 1,共计7. 5 μ 1 ;将以上成分充分混勻、离心后进行反转录(RT),RT参数设置为:16°C 30min,42°C 30min,再以85°C 5min灭活反转录酶,合成的cDNA置_20°C冻存 [0012] Reverse transcription system comprising: a reverse transcriptase 0.5μ 1, reverse transcription buffer 0.75μ 1, RNA inhibitors 0. 095 μ 1, hsa-miR-16 primers lotus ring structure 1. 5 μ 1 , deionized water after 2.08 μ l.dNTP mix 0. 075 μ 1, and digested RNA samples 2. 5 μ 1, a total of 7. 5 μ 1; the above ingredients are thoroughly mixed, after centrifugation for reverse transcription ( RT), RT parameter is set to: 16 ° C 30min, 42 ° C 30min, and then at 85 ° C 5min to inactivate reverse transcriptase, the cDNA synthesis frozen opposing _20 ° C

[0013] (3) hsa-miR-16实时荧光定量PCR的检测 [0013] (3) hsa-miR-16 real-time PCR detection

[0014] 扩增系统包括:含Taq酶的miRNA表达定量检测混合液IOul、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液(该混合液包含20 X扩增引物和Taqman MGB (MinorGroove Binder, 一种非荧光淬灭基因)标记的探针)Iul (上述两种试剂均购于美国ABI公司,USA)和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液Iul和TOsnRNA 扩增引物与U6snRNA荧光标记探针混合液lul。 [0014] The amplification system comprising: containing Taq enzyme mixture quantitative detection of miRNA expression IOul, hsa-miR-16 and the amplification primer hsa-miR-16 a mixture of fluorescently labeled probes (20 X the mixture comprising amplification primer and Taqman MGB (MinorGroove Binder, a non-fluorescent quenching gene) labeled probe) IUL (both reagents were purchased from ABI company, USA) and TOsnRNA TOsnRNA amplification primer with a mixture of fluorescently labeled probes and IUL TOsnRNA U6snRNA amplification primer and fluorescently labeled probe mixture lul. 混合以上成分,加入1.33ul的反转录产物和7. 67ul的去离子水;利用ABI 7300系统进行miRNA定量检测,循环参数设定为95°C IOmin 活化Taq酶,然后95°C 15sec,60°C lmin,共40个循环;选用TOsnRNA为内参,U6snRNA的检测使用与hsa-miR-16相同的反转录和荧光定量PCR反应体系以及TOsnRNA的引物与探针混合液; Mixing the above ingredients, adding 1.33ul reverse transcription product and 7. 67ul of deionized water; using the ABI 7300 system miRNA quantitative detection cycle parameter is set to 95 ° C IOmin activation of Taq polymerase, and 95 ° C 15sec, 60 ° C lmin, 40 cycles; TOsnRNA chosen as internal control, using the same detection U6snRNA hsa-miR-16 reverse transcription and quantitative PCR reaction system and TOsnRNA mixture of primers and probes;

[0015] (4)hsa-miR-16脓毒症患者中表达情况分析 (4) expression of hsa-miR-16 in sepsis [0015] Analysis

[0016] 通过与内参TOsnRNA和正常人的血清含量标准化后计算的2_ΔΔ"水平来计算血清中hsa-miR-16的表达水平,凡是表达水平< 1的患者在28内病死几率大,而表达水平> 1的患者观天病死几率小。 [0016] 2_ΔΔ "level after normalization by the internal reference serum levels TOsnRNA and calculates the calculated normal serum levels of hsa-miR-16, the expression levels of all <1 patient died within 28 large probability, while the expression level of > 1 day outlook patient died little chance.

[0017] 本发明通过以下的方法证明确定了hsa-miR-16为早期预测脓毒症病死率的分子标志物。 [0017] The present invention is demonstrated by the following method of determining the hsa-miR-16 for the early prediction of mortality in sepsis molecular markers.

[0018] 研究对象的选择 Select [0018] object of study

[0019] 本研究选用的研究对象来自于解放军总医院SI⑶、RI⑶和EI⑶病房的脓毒症患者,采血时间为入住ICUM小时内。 [0019] In this study, the subjects from the People's Liberation Army General Hospital SI⑶, sepsis patients RI⑶ and EI⑶ ward, blood collection time to stay ICUM within hours. 同时记录每位患者入院时生病体征及各项化验资料。 At the same time recording the ill signs and laboratory data when each patient on admission. 按照入住ICU的28天病死率分为生存组与死亡组,生存组42例,死亡组34例。 According to the ICU mortality 28 days were divided into groups and death group survival, survival 42 cases, 34 cases died.

[0020] 1.病例血清样本处理及RNA提取[0021] 以促凝管采集促凝血,临床常规血清标本以带分离胶的菲可替管采集。 [0020] 1. Case of serum samples and RNA extraction [0021] In procoagulant coagulant collection tube, Ficoll to routine clinical serum samples with gel separator for collection tube. 参考相关文献获得无细胞血清或血浆,简言之:在4°C下以3000rpm离心15min,将上层血清转移至洁净Ep管,再在4°C下以15,OOOrpm离心30min,以去除细胞碎片,吸取上层血清于洁净离心管,-80°C冻存备用。 Reference literature to obtain a cell-free serum or plasma, in short: at 4 ° C for centrifugation at 3000rpm 15min, the upper layer of serum was transferred to a clean tube Ep, then to 15, OOOrpm centrifuged at 4 ° C for 30min, to remove cell debris , to draw the upper serum clean centrifuge tube, -80 ° C frozen spare. 按照mirVana PARIS试剂盒使用说明书从血清中提取总RNA,使用Scandrop测定RNA提取液的A260吸光度值和A260/A^0的比值(1. 9-2. 1)。 According mirVana PARIS Kit Manual Using Scandrop extract RNA assay A260 absorbance values ​​and A260 / A ^ 0 the ratio (1. 9-2. 1) extracting total RNA from the serum. 变性琼脂糖凝胶电泳检测RNA的完整性。 Integrity denaturing agarose gel electrophoresis of RNA. 然后参照DNase I使用说明书,于37°C对RNA提取液消化30min。 Referring then Manual DNase I at 37 ° C to extract RNA digestion 30min. 于_80°C保存备用。 At _80 ° C for use.

[0022] 2. Hsa-miR-16cDNA 的合成 [0022] 2. Synthesis of the Hsa-miR-16cDNA

[0023] 反转录系统包括:反转录酶0.5μ 1、反转录缓冲液0.75μ 1、RNA酶抑制剂0. 095 μ l、hsa-miR-16 莲环结构引物1. 5 μ 1、去离子水2. 08 μ l.dNTP mix 0. 075 μ 1、及消化后RNA样品2. 5 μ 1,共计7. 5 μ 1。 [0023] Reverse transcription system comprising: a reverse transcriptase 0.5μ 1, reverse transcription buffer 0.75μ 1, RNA inhibitors 0. 095 μ l, hsa-miR-16 primers lotus ring structure 1. 5 μ 1 , deionized water after 2.08 μ l.dNTP mix 0. 075 μ 1, and digested RNA samples 2. 5 μ 1, a total of 7. 5 μ 1. 上述所有试剂均来源于Taqman microRNA反转录试剂盒(Applied Biosytems 公司,USA)。 All of the above reagents were from Taqman microRNA reverse transcription kit (Applied Biosytems Company, USA).

[0024] 将以上成分充分混勻,离心后进行反转录(RT)。 [0024] The above ingredients are thoroughly mixed, reverse transcription (RT) after centrifugation. RT参数设置为:16°C 30min, 42°C 30min,再以85°C 5min灭活反转录酶,合成的cDNA产物置_20°C冻存备用。 RT parameter is set to: 16 ° C 30min, 42 ° C 30min, and then at 85 ° C 5min to inactivate reverse transcriptase, the cDNA synthesis product was frozen spare opposing _20 ° C.

[0025] 3. Hsa-miR-16实时荧光定量PCR的检测 [0025] 3. Hsa-miR-16 real-time PCR detection

[0026] 扩增系统包括:含Taq酶的miRNA表达定量检测混合液IOul、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液(该混合液包含20 X扩增引物和Taqman MGB (MinorGroove Binder,一种非荧光淬灭基因)标记的探针)Iul (上述两种试剂均购于美国ABI公司,USA)和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液Iul和TOsnRNA 扩增引物与U6snRNA荧光标记探针混合液Iul。 [0026] The amplification system comprising: containing Taq enzyme mixture quantitative detection of miRNA expression IOul, hsa-miR-16 and the amplification primer hsa-miR-16 a mixture of fluorescently labeled probes (20 X the mixture comprising amplification primer and Taqman MGB (MinorGroove Binder, a non-fluorescent quenching gene) labeled probe) IUL (both reagents were purchased from ABI company, USA) and TOsnRNA TOsnRNA amplification primer with a mixture of fluorescently labeled probes and IUL TOsnRNA U6snRNA amplification primer and fluorescently labeled probe mixture Iul.

[0027] 将以上成分混勻,再加入1. 33ul的cDNA产物和7. 67ul的去离子水,充分混勻。 [0027] The above components are mixed, then was added and the cDNA product 1. 33ul 7. 67ul of deionized water, and mix thoroughly. 利用ABI7300系统进行miRNA定量检测。 For quantitative detection of miRNA using ABI7300 system. 循环参数设定为95°C lOmin,然后95°C 15sec, 60°C lmin,共40个循环。 Cycling parameters set at 95 ° C lOmin, then 95 ° C 15sec, 60 ° C lmin, 40 cycles. 实验选用TOsnRNA为内参,U6snRNA的检测使用与hsa-miR-16相同的反转录和扩增体系,仅引物与探针使用TOsnRNA的。 TOsnRNA experiment, as internal control, using the same detector hsa-miR-16 U6snRNA reverse transcription and amplification system, only the primers and probes used in TOsnRNA. 反应后记录初始的Ct值。 After the initial reaction Ct values ​​recorded.

[0028] 4. hsa-miR-16预测早期脓毒症患者病死率的情况分析 [0028] 4. The case hsa-miR-16 predicted early mortality in patients with sepsis Analysis

[0029] 通过与内参TOsnRNA和正常人的血清含量标准化后计算的2_ΔΔ"水平来计算血清中hsa-miR-16的表达水平,凡是表达水平< 1的患者在28内病死几率大,而表达水平> 1的患者观天病死几率小。 [0029] 2_ΔΔ "level after normalization by the internal reference serum levels TOsnRNA and calculates the calculated normal serum levels of hsa-miR-16, the expression levels of all <1 patient died within 28 large probability, while the expression level of > 1 day outlook patient died little chance.

附图说明 BRIEF DESCRIPTION

[0030] 图1为hsa-miR-16在正常组(NC),脓毒症患者生存组(S)和死亡组(D)血清中表达水平比较。 [0030] Figure 1 is hsa-miR-16 in the normal group (NC), the expression levels of survival in sepsis patients (S) and death group (D) Comparison of serum. 图中生存组表达水平高于正常组,而死亡组表达水平低于正常组。 Figure expression levels in survival group than the normal group, the expression level of the group of death than normal group.

[0031] 图2为hsa-miR-16,hsa_miR-15b,hsa-miR-223同已有的评价脓毒症预后指标CRP (C-反应蛋白),PCT (降钙素原),APECHE II评分(急性生理与慢性健康评分)和SOFA 评分(感染相关的器官衰竭评分)预测预后价值比较的ROC(受试者工作曲线)曲线,具体数值见表1所示。 [0031] Figure 2 is hsa-miR-16, hsa_miR-15b, hsa-miR-223 with the existing evaluation sepsis prognostic markers CRP (C- reactive protein), PCT (procalcitonin), APECHE II score (acute physiology and chronic health evaluation) and SOFA score (infection-related organ failure assessment) prognostic value compared ROC (receiver operating curve) curve, the values ​​shown in Table 1.

[0032] 参见附图,hsa-miR-16的表达水平以2_ΔΔα表示,-AACt = (Ct脓毒症患^-CtJ-(Ct ^a-CtJ0图中显示的表达量是经过与U6snRNA标准化后的表达量。由图1 可以看出,死亡组的hsa-miR-16表达水平低于正常组,而生存组hsa-miR-16表达水平高于正常组。而生存组与死亡组之间通过t检验,ρ = 0.009,差异有统计学意义。通过One-way ANOVA分析后ρ = 0.017,说明三组之间总体均值是有差异的。 [0032] Referring to the drawings, the expression levels of hsa-miR-16 is expressed in 2_ΔΔα, -AACt = (Ct sepsis patients ^ -CtJ- (expression amount shown in FIG Ct ^ a-CtJ0 is elapsed after normalization with U6snRNA the expression level can be seen from Figure 1, hsa-miR-16 expression level higher than the normal death group, survival group hsa-miR-16 expression level higher than the normal group. through between survival and death group group t-test, ρ = 0.009, statistically significant differences by One-way ANOVA analysis after ρ = 0.017, describes the overall mean of the three groups is different.

[0033] 上述数据说明hsa-miR-16的表达水平可以用来预测早期脓毒症患者的28天病死率。 [0033] The above data demonstrate the expression levels of hsa-miR-16 may be used to predict early 28-day mortality in patients with sepsis.

[0034] 为进一步分析,我们选取的同脓毒症相关的miRNAs和常用于脓毒症预后评价的指标绘制ROC曲线,并且比较它们的曲线下面积(AUC),结果显示hsa-miR-16有最大的曲线下面积0. 847,ρ < 0. 001,说明其预测脓毒症患者病死率的价值最大,如表1和图2中所不。 [0034] For further analysis, we selected miRNAs associated with sepsis and sepsis used to evaluate the prognostic indicator plotted ROC curve, and comparing the area under the curve thereof (AUC), results showed that there is hsa-miR-16 largest area under the curve 0. 847, ρ <0. 001, a maximum value described prediction of mortality in patients with sepsis, as shown in table 1 and FIG. 2 is not.

[0035] 表1. miRNAs, CRP, PCT, SOFA评分和APECHE II评分的曲线下面积 Area [0035] Table 1. miRNAs, CRP, PCT, SOFA score and APECHE II score curve

[0036] [0036]

Figure CN102191327AD00061

[0037] # :APACHE II评分指急性生理与慢性健康评分;SOFA评分指感染相关器官衰竭评分系统;CRP =C反应蛋白;PCT :降钙素原。 [0037] #: APACHE II score refers to acute physiology and chronic health evaluation; SOFA score refers to an infection associated system organ failure rates; CRP = C-reactive protein; PCT: Procalcitonin.

[0038] 将与脓毒症相关的指标包括性别,年龄,APECHE II评分,CRP, PCT, SOFA评分, hsa-miR-223, hsa_miR-15b,hsa-miR-16, WBC (白细胞),体温以及伴随疾病(高血压,糖尿病,慢性阻塞性肺病,冠心病和免疫抑制)纳入二分类变量单因素回归分析(如表2所示)。 [0038] The index associated with sepsis including gender, age, APECHE II scores, CRP, PCT, SOFA score, hsa-miR-223, hsa_miR-15b, hsa-miR-16, WBC (white blood cells), and body temperature univariate comorbidities (hypertension, diabetes, chronic obstructive pulmonary disease, coronary artery disease, and immunosuppression) into binary regression analysis (table 2).

[0039] 表2.生存组与死亡组之间的二分类变量单因素logistic回归分析 [0039] dichotomous Table 2. Univariate survival between the group and the death group logistic regression analysis

[0040] [0040]

Figure CN102191327AD00062

[0041] [0041]

Figure CN102191327AD00071

[0042] 将ρ < 0. 05的变量纳入二分类变量的多因素分析后,结果只有APECHE II评分, SOFA评分,hsa-miR-223和hsa_miR-16进入回归方程(如表3所示),hsa-miR-16的相对危险度(OR)值为0. 135,说明hsa-miR-16是脓毒症的一个独立的保护因素,与上述t检验 After Multivariate analysis [0042] The variable ρ <0.05 into two categorical variables, the result is only APECHE II score, SOFA score, hsa-miR-223, and into the regression equation hsa_miR-16 (Table 3), the relative risk of hsa-miR-16 (OR) is 0.135, indicating that hsa-miR-16 is an independent protective factor sepsis, as in the t-test

结果一致。 Results are consistent.

[0043] 表3. 二分类变量多因素logistic回归分析结果 [0043] Table 3. dichotomous multivariate logistic regression analysis

[0044] [0044]

Figure CN102191327AD00072

[0045] 本发明首次利用miRNAs做为一个生物标记物评价脓毒症的预后。 [0045] The present invention is the first use of miRNAs as a prognostic biomarker for sepsis. 首次在脓毒症患者血清中发现hsa-miR-16同脓毒症预后的相关性。 First found hsa-miR-16 with the prognosis of sepsis in the patients with sepsis. 进一步验证hsa-miR-16与脓毒症病死率的相关性,并与临床相关指标结合,为评估脓毒症患者预后提供可信的分子标志物。 Further validate the correlation hsa-miR-16 mortality associated with sepsis, and combined with the clinically relevant indicators provide reliable prognostic molecular markers for evaluating sepsis patients. 所提供的试剂盒及其步骤对血清中miRNAs的检测和定量准确可靠。 Kit and its step provides accurate and reliable detection and quantitation of miRNAs in serum.

[0046] 本发明的优点为:(1)本发明确定的miRNAs与脓毒症预后密切相关,能够为临床个体化干预治疗提供有效的依据。 [0046] The advantages of the present invention are: (1) is closely related to the present invention is identified miRNAs and prognosis of sepsis can be individualized for clinical intervention to provide effective basis. (2)实时荧光定量PCR技术应用与miRNAs的检测与定量,方面实用。 (2) Detection and quantitative real-time PCR and Application of miRNAs, practical aspects. (¾临床样本获取方便,可直接抽取患者血样后获得血清进行检测。(4) hsa-miR-16预测病死率的价值大于常用指标SOFA评分与APECHE II评分,并且可与上述指标结合,为脓毒症预后的评估提供综合性的试剂盒,方便于临床应用。 具体实施方式 (¾ convenient to obtain clinical specimens, blood samples may be drawn directly obtain patient sera were tested after (4) the value of hsa-miR-16 predicted mortality and greater than a commonly used indicator SOFA score APECHE II score, and may be combined with the index, pus thyrotoxicosis prognostic evaluation kits provide comprehensive, convenient for clinical application. DETAILED DESCRIPTION

[0047] 下面将结合具体例子对本发明的技术方案做更详细的说明,这些例子仅仅是用于说明本发明的技术构思和实现方式的,而不是用于限定本发明的范围。 [0047] Specific examples will now be made in conjunction with a more detailed description of the technical solution of the present invention, these examples are merely to illustrate the technical concepts and implementations of the present invention, and not intended to limit the scope of the invention.

[0048] 实施例1 [0048] Example 1

[0049] 一种用于预测脓毒症患者病死率的试剂盒,该试剂盒由反转录系统和扩增系统组成,其中,反转录系统由反转录酶、反转录体系缓冲液、RNA酶抑制剂、dNTP mix,hsa-miR-16 茎环结构引物和消化后的RNA提取液组成,扩增系统由含Taq酶的miRNA表达定量检测混合液、hsa-miR-16扩增引物与hsa_miR-16荧光标记探针混合液和TOsnRNA扩增引物与UBsnRNA荧光标记探针混合液组成。 [0049] A method for prediction of mortality in patients with sepsis kits, the kit was amplified by the Reverse Transcription System and the system components, wherein the Reverse Transcription System by the reverse transcriptase, reverse transcriptase buffer system , RNA inhibitors, dNTP mix, hsa-miR-16 stem-loop structure after digestion of the primers and RNA extraction solution composition, the amplification system containing miRNA expression quantitative detection of Taq enzyme mixture, hsa-miR-16 amplification primer hsa_miR-16 with a mixture of fluorescently labeled probes and amplification primers UBsnRNA TOsnRNA fluorescently labeled probe mixture solution.

[0050] 应用该试剂盒来早期预测脓毒症患者病死率的具体方法如下: [0051 ] 1.病例血清样本处理及RNA提取 [0050] The method of application of the kit specific mortality in septic patients early prediction to the following: [0051] 1. Case of serum samples RNA extraction and

[0052] 以促凝管采集促凝血,临床常规血清标本以带分离胶的菲可替管采集。 [0052] In procoagulant coagulant collection tube, in routine clinical serum specimens with Ficoll gel separator for collection tube. 抽取入住ICU8例脓毒症患者M小时内的血样以及4正常人血样。 Check the blood drawn within hours of M 4 ICU8 patients with sepsis and healthy blood. 在4°C下以3000rpm离心15min,将上层血清转移至洁净Ep管,再在4°C下以15,OOOrpm离心30min,以去除细胞碎片,吸取上层血清于洁净离心管,-80°C冻存备用。 At 4 ° C for centrifugation at 3000rpm 15min, the upper layer of serum was transferred to a clean tube Ep, then to 15, OOOrpm centrifuged at 4 ° C for 30min, to remove cell debris, the serum to draw the upper clean centrifuge tube, -80 ° C frozen memory backup. 按照mirVana PARIS试剂盒使用说明书从血清中提取总RNA,使用kandrop测定RNA提取液的A260吸光度值和A260/A280的比值(1. 9-2. 1) 以评估其浓度和治疗。 MirVana PARIS Kit according to the instructions for use Total RNA was extracted from serum using a RNA extraction was measured kandrop A260 absorbance values ​​and the ratio of A260 / A280 of (1. 9-2. 1) to assess the concentration and treatment. 变性琼脂糖凝胶电泳检测RNA的完整性。 Integrity denaturing agarose gel electrophoresis of RNA. 然后参照DNase I使用说明书,于37°C对RNA提取液消化30min。 Referring then Manual DNase I at 37 ° C to extract RNA digestion 30min. 于_80°C保存备用。 At _80 ° C for use.

[0053] 2. Hsa-miR-16cDNA 的合成 [0053] 2. Synthesis of the Hsa-miR-16cDNA

[0054] 反转录体系包括:反转录酶0.5μ 1、反转录缓冲液0.75μ 1、RNA酶抑制剂0. 095 μ 1,hsa-miR-16 莲环结构引物1. 5 μ 1、去离子水2. 08 μ l.dNTP mix 0. 075 μ 1、及消化后RNA样品2. 5 μ 1,共计7. 5 μ 1。 [0054] Reverse transcription system comprising: a reverse transcriptase 0.5μ 1, reverse transcription buffer 0.75μ 1, RNA inhibitors 0. 095 μ 1, hsa-miR-16 primers lotus ring structure 1. 5 μ 1 , deionized water after 2.08 μ l.dNTP mix 0. 075 μ 1, and digested RNA samples 2. 5 μ 1, a total of 7. 5 μ 1. 充分混勻、离心后进行反转录(RT)。 Mix well, reverse transcription (RT) after centrifugation. RT参数设置为: 160C 30min,42°C 30min,再以85°C 5min灭活反转录酶,合成的cDNA置_20°C冻存备用。 RT parameter set: 160C 30min, 42 ° C 30min, and then at 85 ° C 5min to inactivate reverse transcriptase, the cDNA synthesis frozen spare opposing _20 ° C. 上述所有试剂均来源于iTaqman microRNA反转录试剂盒(Applied Biosytems公司,USA)。 All of the above reagents were from iTaqman microRNA reverse transcription kit (Applied Biosytems Company, USA).

[0055] 3. Hsa-miR-16实时荧光定量PCR的检测 [0055] 3. Hsa-miR-16 real-time PCR detection

[0056] 扩增系统包括:含Taq酶的miRNA表达定量检测混合液IOul、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液(该混合液包含20 X扩增引物和Taqman MGB (MinorGroove Binder,一种非荧光淬灭基因)标记的探针)Iul (上述两种试剂均购于美国ABI公司,USA)和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液Iul和TOsnRNA 扩增引物与TOsnRNA荧光标记探针混合液lul。 [0056] The amplification system comprising: containing Taq enzyme mixture quantitative detection of miRNA expression IOul, hsa-miR-16 and the amplification primer hsa-miR-16 a mixture of fluorescently labeled probes (20 X the mixture comprising amplification primer and Taqman MGB (MinorGroove Binder, a non-fluorescent quenching gene) labeled probe) IUL (both reagents were purchased from ABI company, USA) and TOsnRNA TOsnRNA amplification primer with a mixture of fluorescently labeled probes and IUL TOsnRNA TOsnRNA amplification primer and fluorescently labeled probe mixture lul. hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液(该混合液包含20 X扩增引物和Taqman MGB (Minor Groove Binder,一种非荧光淬灭基因)标记的探针)Iul (上述两种试剂均购于美国ABI公司,USA)和TOsnRNA 扩增引物与TOsnRNA荧光标记探针混合液Iul和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液lul。 hsa-miR-16 and the amplification primer hsa-miR-16 a mixture of fluorescently labeled probes (20 X the mixture comprising amplification primers and Taqman MGB (Minor Groove Binder, a non-fluorescent quenching gene) probes labeled ) IUL (both reagents were purchased from ABI company, USA) and TOsnRNA TOsnRNA amplification primer with a mixture of fluorescently labeled probes and TOsnRNA IUL TOsnRNA amplification primer and fluorescently labeled probe mixture lul. hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液(该混合液包含20 X扩增引物和iTaqman MGB (Minor Groove Binder,一种非荧光淬灭基因)标记的探针)lul (上述两种试剂均购于美国ABI公司,USA)和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液Iul和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液lul。 hsa-miR-16 and the amplification primer hsa-miR-16 a mixture of fluorescently labeled probes (20 X the mixture comprises primers and amplification probes iTaqman MGB (Minor Groove Binder, a non-fluorescent quenching gene) marked ) LUL (both reagents were purchased from ABI company, USA) and TOsnRNA amplification primer and fluorescent probe mixture Iul TOsnRNA TOsnRNA amplification primer and the fluorescently labeled probe mixture TOsnRNA lul. 混合以上成分,加入1. 33ul的反转录产物和7. 67ul的去离子水。 Mixing the above ingredients, adding 1. 33ul of the reverse transcribed product and 7. 67ul of deionized water. 利用ABI7300系统进行miRNA定量检测。 For quantitative detection of miRNA using ABI7300 system. 循环参数设定为95°C IOmin活化Taq酶,然后95°C 15sec,60°C lmin,共40个循环。 Cycling parameters set at 95 ° C IOmin activation of Taq polymerase, and 95 ° C 15sec, 60 ° C lmin, 40 cycles. 实验选用TOsnRNA为内参,TOsnRNA的检测使用与hsa_miR-16相同的反转录和荧光定量PCR 反应体系以及TOsnRNA的引物与探针系统。 TOsnRNA experiment, as internal control, using the same detector and TOsnRNA hsa_miR-16 reverse transcription and quantitative PCR reaction system and the primers and probes TOsnRNA system.

[0057] 4. hsa-miR-16在8例脓毒症患者中表达情况分析 [0057] 4. hsa-miR-16 expression analysis in patients with sepsis 8

[0058] 通过与内参TOsnRNA和正常人的血清含量标准化后计算的2_ΔΔενΚ平来计算血清中hsa-miR-16的表达水平,凡是表达水平< 1的患者在28内病死几率大,而表达水平> 1 的患者28天病死几率小。 [0058] The calculated expression levels in serum hsa-miR-16 level by 2_ΔΔενΚ calculated after normalization with the internal reference serum levels and normal TOsnRNA, any level of expression of <1 patient died within 28 large probability, while the expression level of> 1 28-day mortality in patients with little chance. 本研究中8例患者以28天病死率算,最终6例生存,两例死亡,6 例生存患者经标准化后的表达水平分别为7. 342,9. 021,3. 890,2. 172,4. 214和1. 593。 In this study, 8 patients of 28 day mortality count, eventually survive 6 cases, two deaths, the expression level of normalized survival after 6 patients respectively 7. 342,9. 021,3. 890,2. 172, 4.214 and 1.593. 两例死亡患者的表达水平为0. 629和0. 143,两组差异有统计学意义,且生存患者hsa-miR-16 的表达水平均大于1,而死亡组的表达量均小于,与本研究结果符合。 The expression levels for two deaths in patients 0.629 and 0.143, showing significant difference in the survival and expression levels of hsa-miR-16 in patients of average of greater than 1, and expression levels of less than death group, this The results accord. 然而该6例生存患者入院的APACHE II评分分别是24,16,21,5,19和21,死亡患者的APACHE II评分是M和27,差异无统计学意义,且不能明确预测患者病死率。 However, the survival of patients admitted to hospital six cases of APACHE II scores were 24,16,21,5,19 and 21, the death of patients APACHE II score was 27 and M, was not statistically significant, and can not clearly predict patient mortality. 说明Hsa-miR-16在预测脓毒症患者病死率方面的价值要优于常用指标APACHE II评分。 Description Hsa-miR-16 in terms of predictive value for mortality in patients with sepsis is superior to commonly used indicators of APACHE II score. 该实例证实了该试剂盒的实用性。 This example demonstrates the utility of the kit.

[0059] 实施例2 [0059] Example 2

[0060] 一种用于预测脓毒症患者病死率的试剂盒,该试剂盒由反转录系统和扩增系统组成,其中,反转录系统由反转录酶、反转录体系缓冲液、RNA酶抑制剂、dNTP mix,hsa-miR-16 茎环结构引物和消化后的RNA提取液组成,扩增系统由含Taq酶的miRNA表达定量检测混合液、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液和TOsnRNA扩增引物与UBsnRNA荧光标记探针混合液组成。 [0060] A method for prediction of mortality in patients with sepsis kits, the kit was amplified by the Reverse Transcription System and the system components, wherein the Reverse Transcription System by the reverse transcriptase, reverse transcriptase buffer system , RNA inhibitors, dNTP mix, hsa-miR-16 stem-loop structure after digestion of the primers and RNA extraction solution composition, the amplification system containing miRNA expression quantitative detection of Taq enzyme mixture, hsa-miR-16 amplification primer and hsa-miR-16 a mixture of fluorescently labeled probes and amplification primers UBsnRNA TOsnRNA fluorescently labeled probe mixture solution.

[0061] 应用该试剂盒来早期预测脓毒症患者病死率的具体方法如下: [0061] The method of application of the kit specific mortality in septic patients early prediction for the following:

[0062] 1.病例血清样本处理及RNA提取 [0062] 1. Case of serum samples RNA extraction and

[0063] 以促凝管采集促凝血,临床常规血清标本以带分离胶的菲可替管采集。 [0063] In procoagulant coagulant collection tube, in routine clinical serum specimens with Ficoll gel separator for collection tube. 抽取入住I⑶10例脓毒症患者M小时内的血样。 Check the blood sample drawn in patients with sepsis M I⑶10 hours. 在4°C下以3000rpm离心15min,将上层血清转移至洁净Ep管,再在4°C下以15,OOOrpm离心30min,以去除细胞碎片,吸取上层血清于洁净离心管,-80°C冻存备用。 At 4 ° C for centrifugation at 3000rpm 15min, the upper layer of serum was transferred to a clean tube Ep, then to 15, OOOrpm centrifuged at 4 ° C for 30min, to remove cell debris, the serum to draw the upper clean centrifuge tube, -80 ° C frozen memory backup. 按照mirVana PARIS试剂盒使用说明书从血清中提取总RNA,使用Scandrop测定RNA提取液的A260吸光度值和A260/A^0的比值(1. 9-2. 1)以评估其浓度和治疗。 MirVana PARIS Kit according to the instructions for use Total RNA was extracted from serum using a RNA extraction solution Scandrop measured absorbance values ​​A260 and A260 / A ^ 0 the ratio (1. 9-2. 1) to assess the concentration and treatment. 变性琼脂糖凝胶电泳检测RNA的完整性。 Integrity denaturing agarose gel electrophoresis of RNA. 然后参照DNase I使用说明书,于37°C 对RNA提取液消化30min。 Referring then Manual DNase I at 37 ° C to extract RNA digestion 30min. 于_80°C保存备用。 At _80 ° C for use.

[0064] 2. Hsa-miR-16cDNA 的合成 [0064] 2. Synthesis of the Hsa-miR-16cDNA

[0065] 反转录体系包括:反转录酶0.5μ 1、反转录缓冲液0.75μ 1、RNA酶抑制剂0. 095 μ 1,hsa-miR-16 莲环结构引物1. 5 μ 1、去离子水2. 08 μ l.dNTP mix 0. 075 μ 1、及消化后RNA样品2. 5 μ 1,共计7. 5 μ 1。 [0065] Reverse transcription system comprising: a reverse transcriptase 0.5μ 1, reverse transcription buffer 0.75μ 1, RNA inhibitors 0. 095 μ 1, hsa-miR-16 primers lotus ring structure 1. 5 μ 1 , deionized water after 2.08 μ l.dNTP mix 0. 075 μ 1, and digested RNA samples 2. 5 μ 1, a total of 7. 5 μ 1. 充分混勻、离心后进行反转录(RT)。 Mix well, reverse transcription (RT) after centrifugation. RT参数设置为: 160C 30min,42°C 30min,再以85°C 5min灭活反转录酶,合成的cDNA置_20°C冻存备用。 RT parameter set: 160C 30min, 42 ° C 30min, and then at 85 ° C 5min to inactivate reverse transcriptase, the cDNA synthesis frozen spare opposing _20 ° C. 上述所有试剂均来源于iTaqman microRNA反转录试剂盒(Applied Biosytems公司,USA)。 All of the above reagents were from iTaqman microRNA reverse transcription kit (Applied Biosytems Company, USA).

[0066] 3. Hsa-miR-16实时荧光定量PCR的检测 [0066] 3. Hsa-miR-16 real-time PCR detection

[0067] 扩增系统包括:含Taq酶的miRNA表达定量检测混合液IOul、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液(该混合液包含20 X扩增引物和Taqman MGB (MinorGroove Binder, 一种非荧光淬灭基因)标记的探针)Iul (上述两种试剂均购于美国ABI公司,USA)和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液Iul和TOsnRNA 扩增引物与U6snRNA荧光标记探针混合液lul。 [0067] The amplification system comprising: containing Taq enzyme mixture quantitative detection of miRNA expression IOul, hsa-miR-16 and the amplification primer hsa-miR-16 a mixture of fluorescently labeled probes (20 X the mixture comprising amplification primer and Taqman MGB (MinorGroove Binder, a non-fluorescent quenching gene) labeled probe) IUL (both reagents were purchased from ABI company, USA) and TOsnRNA TOsnRNA amplification primer with a mixture of fluorescently labeled probes and IUL TOsnRNA U6snRNA amplification primer and fluorescently labeled probe mixture lul. 混合以上成分,加入1.33ul的反转录产物和7. 67ul的去离子水。 Mixing the above ingredients, adding 1.33ul reverse transcription product and 7. 67ul of deionized water. 利用ABI7300系统进行miRNA定量检测。 For quantitative detection of miRNA using ABI7300 system. 循环参数设定为95°C IOmin 活化Taq酶,然后95°C 15sec,60°C lmin,共40个循环。 Cycling parameters set at 95 ° C IOmin activation of Taq polymerase, and 95 ° C 15sec, 60 ° C lmin, 40 cycles. 实验选用TOsnRNA为内参,U6snRNA 的检测使用与hsa-miR-16相同的反转录和荧光定量PCR反应体系以及TOsnRNA的引物与探针系统。 TOsnRNA experiment, as internal control, using the same detection U6snRNA and hsa-miR-16 reverse transcription and quantitative PCR reaction system and the primers and probes TOsnRNA system.

[0068] 4. hsa-miR-16在10例脓毒症患者中表达情况分析 [0068] 4. hsa-miR-16 expression in 10 cases of patients with sepsis Analysis

[0069] 通过与内参TOsnRNA和正常人的血清(仍采用上实施例中正常人的表达水平)含量标准化后计算的2_ΔΔα水平来计算血清中hsa-miR-16的表达水平,凡是表达水平< 1 的患者在28内病死几率大,而表达水平> 1的患者28天病死几率小。 [0069] By TOsnRNA internal reference serum and normal (still using normal level of expression of the embodiment of the embodiment) 2_ΔΔα level calculation to calculate the content of the normalized expression levels in serum hsa-miR-16, the expression levels of all <1 the patient died within 28 big chance, and the expression levels of> 1 patient died 28 days small chance. 本研究中10例患者以观天病死率算,最终7例均生存,3例患者死亡。 In this study, 10 patients with Sky fatality count, the final seven cases are to survive, three patients died. 7例患者的经标准化后的表达水平分别为3. 970,13. 576,5. 342,1. 453,7. 001,2. 175 和8. 011,表达量均大于1。3 例死亡患者hsa-miR-16的表达水平为0. 218,0. 928和0. 190,均小于1,并且两组差异有统计学意义,符合本研究结果。 Normalized expression levels of the 7 patients, respectively 3. 970,13. 576,5. 342,1. 453,7. 001,2. 175 and 8.011, expression levels greater than 1.3 deaths in patients the expression level of hsa-miR-16 0. 218,0. 928 and 0.190, less than 1, and the difference was statistically significant, consistent with the present findings. 说明hsa-miR-16经标准化后的表达水平可以很好的预测脓毒症患者的28 天病死率。 Description hsa-miR-16 expression level was normalized perfect predictor of 28-day mortality in patients with sepsis.

[0070] 实施例3 [0070] Example 3

[0071] 一种用于预测脓毒症患者病死率的试剂盒,该试剂盒由反转录系统和扩增系统组成,其中,反转录系统由反转录酶、反转录体系缓冲液、RNA酶抑制剂、dNTP mix,hsa-miR-16 茎环结构引物和消化后的RNA提取液组成,扩增系统由含Taq酶的miRNA表达定量检测混合液、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液和TOsnRNA扩增引物与UBsnRNA荧光标记探针混合液组成。 [0071] A method for prediction of mortality in patients with sepsis kits, the kit was amplified by the Reverse Transcription System and the system components, wherein the Reverse Transcription System by the reverse transcriptase, reverse transcriptase buffer system , RNA inhibitors, dNTP mix, hsa-miR-16 stem-loop structure after digestion of the primers and RNA extraction solution composition, the amplification system containing miRNA expression quantitative detection of Taq enzyme mixture, hsa-miR-16 amplification primer and hsa-miR-16 a mixture of fluorescently labeled probes and amplification primers UBsnRNA TOsnRNA fluorescently labeled probe mixture solution.

[0072] 应用该试剂盒来早期预测脓毒症患者病死率的具体方法如下: [0072] The method of application of the kit specific mortality in septic patients early prediction for the following:

[0073] 1.病例血清样本处理及RNA提取 [0073] 1. Case of serum samples RNA extraction and

[0074] 以促凝管采集促凝血,临床常规血清标本以带分离胶的菲可替管采集。 [0074] In procoagulant coagulant collection tube, in routine clinical serum specimens with Ficoll gel separator for collection tube. 抽取入住I⑶12例脓毒症患者M小时内的血样。 Check the blood sample drawn in patients with sepsis M I⑶12 hours. 在4°C下以3000rpm离心15min,将上层血清转移至洁净Ep管,再在4°C下以15,OOOrpm离心30min,以去除细胞碎片,吸取上层血清于洁净离心管,-80°C冻存备用。 At 4 ° C for centrifugation at 3000rpm 15min, the upper layer of serum was transferred to a clean tube Ep, then to 15, OOOrpm centrifuged at 4 ° C for 30min, to remove cell debris, the serum to draw the upper clean centrifuge tube, -80 ° C frozen memory backup. 按照mirVana PARIS试剂盒使用说明书从血清中提取总RNA,使用Scandrop测定RNA提取液的A260吸光度值和A260/A^0的比值(1. 9-2. 1)以评估其浓度和治疗。 MirVana PARIS Kit according to the instructions for use Total RNA was extracted from serum using a RNA extraction solution Scandrop measured absorbance values ​​A260 and A260 / A ^ 0 the ratio (1. 9-2. 1) to assess the concentration and treatment. 变性琼脂糖凝胶电泳检测RNA的完整性。 Integrity denaturing agarose gel electrophoresis of RNA. 然后参照DNase I使用说明书,于37°C 对RNA提取液消化30min。 Referring then Manual DNase I at 37 ° C to extract RNA digestion 30min. 于_80°C保存备用。 At _80 ° C for use.

[0075] 2. Hsa-miR-16cDNA 的合成 [0075] 2. Synthesis of the Hsa-miR-16cDNA

[0076] 反转录体系包括:反转录酶0.5μ 1、反转录缓冲液0.75μ 1、RNA酶抑制剂0. 095 μ 1,hsa-miR-16 莲环结构引物1. 5 μ 1、去离子水2. 08 μ l.dNTP mix 0. 075 μ 1、及消化后RNA样品2. 5 μ 1,共计7. 5 μ 1。 [0076] Reverse transcription system comprising: a reverse transcriptase 0.5μ 1, reverse transcription buffer 0.75μ 1, RNA inhibitors 0. 095 μ 1, hsa-miR-16 primers lotus ring structure 1. 5 μ 1 , deionized water after 2.08 μ l.dNTP mix 0. 075 μ 1, and digested RNA samples 2. 5 μ 1, a total of 7. 5 μ 1. 充分混勻、离心后进行反转录(RT)。 Mix well, reverse transcription (RT) after centrifugation. RT参数设置为: 160C 30min,42°C 30min,再以85°C 5min灭活反转录酶,合成的cDNA置_20°C冻存备用。 RT parameter set: 160C 30min, 42 ° C 30min, and then at 85 ° C 5min to inactivate reverse transcriptase, the cDNA synthesis frozen spare opposing _20 ° C. 上述所有试剂均来源于iTaqman microRNA反转录试剂盒(Applied Biosytems公司,USA)。 All of the above reagents were from iTaqman microRNA reverse transcription kit (Applied Biosytems Company, USA).

[0077] 3. Hsa-miR-16实时荧光定量PCR的检测 [0077] 3. Hsa-miR-16 real-time PCR detection

[0078] 扩增系统包括:含Taq酶的miRNA表达定量检测混合液IOul、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液(该混合液包含20 X扩增引物和TaqmanMGB (MinorGroove Binder, 一种非荧光淬灭基因)标记的探针)Iul (上述两种试剂均购于美国ABI公司,USA)和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液Iul和TOsnRNA 扩增引物与U6snRNA荧光标记探针混合液lul。 [0078] The amplification system comprising: containing Taq enzyme mixture quantitative detection of miRNA expression IOul, hsa-miR-16 and the amplification primer hsa-miR-16 a mixture of fluorescently labeled probes (20 X the mixture comprising amplification primer and TaqmanMGB (MinorGroove Binder, a non-fluorescent quenching gene) labeled probe) IUL (both reagents were purchased from ABI company, USA) and TOsnRNA TOsnRNA amplification primer with a mixture of fluorescently labeled probes and TOsnRNA IUL U6snRNA amplification primer and fluorescently labeled probe mixture lul. 混合以上成分,加入1.33ul的反转录产物和7. 67ul的去离子水。 Mixing the above ingredients, adding 1.33ul reverse transcription product and 7. 67ul of deionized water. 利用ABI7300系统进行miRNA定量检测。 For quantitative detection of miRNA using ABI7300 system. 循环参数设定为95°C IOmin 活化Taq酶,然后95°C 15sec,60°C lmin,共40个循环。 Cycling parameters set at 95 ° C IOmin activation of Taq polymerase, and 95 ° C 15sec, 60 ° C lmin, 40 cycles. 实验选用TOsnRNA为内参,U6snRNA 的检测使用与hsa-miR-16相同的反转录和荧光定量PCR反应体系以及TOsnRNA的引物与探针系统。 TOsnRNA experiment, as internal control, using the same detection U6snRNA and hsa-miR-16 reverse transcription and quantitative PCR reaction system and the primers and probes TOsnRNA system.

[0079] 4. hsa-miR-16在12例脓毒症患者中表达情况分析 [0079] 4. hsa-miR-16 expression in 12 cases of patients with sepsis Analysis

[0080] 通过与内参TOsnRNA和正常人的血清含量标准化后计算的2_ΔΔενΚ平来计算血清中hsa-miR-16的表达水平。 [0080] The expression level calculated by the level calculating 2_ΔΔενΚ after normalization with the internal reference serum levels TOsnRNA and normal human serum of hsa-miR-16. 在该研究中,12例患者编号为1111,1112,1113,1114,1115, 1116,1117,1118,1119,11110,IIIll 和11112。 In this study, 12 patients numbered 1111,1112,1113,1114,1115, 1116,1117,1118,1119,11110, IIIll and 11112. 经标准化后的表达水平分别为0. 802, 10. 321,4. 342,0. 231,1. 938,4. 210,2. 191,1. 956,0. 813,20. 023,3. 218,0. 019,最终该12 例患者1111,1114,II19和II112分别死亡于入住ICU的第12天,第3天,第20天和第1 天。 Expression levels were normalized after 0.802, 10 321,4. 342,0. 231,1. 938,4. 210,2. 191,1. 956,0. 813,20. 023,3. 218,0. 019, eventually the 12 patients 1111,1114, II19 and mortality in the ICU II112, respectively on day 12, day 3, and day 20, 1. 实际应用于临床患者后,验证了hsa-miR-16在预测脓毒症患者病死率方面的准确性。 After the actual clinical application in patients, to verify the accuracy of hsa-miR-16 in terms of predicting mortality in patients with sepsis. 并且优于现有的临床常用指标。 And better than existing clinical commonly used indicator.

Claims (6)

  1. 1. 一种用于预测脓毒症患者病死率的试剂盒,其特征在于,该试剂盒由反转录系统和扩增系统组成,其中,反转录系统由反转录酶、反转录体系缓冲液、RNA酶抑制剂、dNTP mix、 hsa-miR-16茎环结构引物和消化后的RNA提取液组成,扩增系统由含Taq酶的miRNA表达定量检测混合液、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液组成。 1. A method for predicting mortality in patients with sepsis kit, wherein the kit by the reverse transcription and amplification system components system, wherein the Reverse Transcription System by the reverse transcriptase, reverse transcriptase buffer system, RNA inhibitor, dNTP mix, hsa-miR-16 stem-loop structure after digestion of the primers and RNA extraction solution composition, quantitative amplification system containing a mixture of Taq polymerase expression of miRNA, hsa-miR-16 amplification primer and hsa-miR-16 a mixture of fluorescently labeled probes and amplification primers TOsnRNA TOsnRNA fluorescently labeled probe mixture solution.
  2. 2.如权利要求1所述的试剂盒,其特征在于所述反转录系统包括:反转录酶0. 5μ 1、反转录缓冲液0. 75μ 1、RNA酶抑制剂0. 095μ l、hsa-miR-16茎环结构引物1. 5 μ 1、去离子水2. 08 μ l.dNTP mix 0. 075 μ 1、及消化后RNA 样品2. 5 μ 1,共计7. 5 μ 1。 2. The kit according to claim 1, wherein said reverse transcription system comprising: a reverse transcriptase 0. 5μ 1, reverse transcription buffer 0. 75μ 1, RNA inhibitors 0. 095μ l , hsa-miR-16 stem-loop primers 1. 5 μ 1, the deionized water 2.08 μ l.dNTP mix 0. 075 μ 1, and digested RNA samples 2. 5 μ 1, 7. 5 μ 1 total .
  3. 3.如权利要求1所述的试剂盒,其特征在于所述扩增系统包括:含Taq酶的miRNA表达定量检测混合液IOul、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液Iul和TOsnRNA扩增引物与TOsnRNA荧光标记探针混合液Iul。 3. The kit according to claim 1, wherein said amplification system comprises: miRNA expression quantitative detection IOul mixture containing Taq enzyme, amplification of hsa-miR-16 primers hsa-miR-16 fluorescent label probe mixture IUL TOsnRNA amplification primer and the fluorescently labeled probe mixture TOsnRNA Iul.
  4. 4. 一种利用上述权利要求中任意一项所述的试剂盒预测脓毒症患者病死率的方法,包括以下步骤:(1)病例血清样本处理及RNA提取以促凝管采集促凝血,临床常规血清标本以带分离胶的菲可替管采集,抽取入住ICU 脓毒症患者M小时内的血样以及正常人血样,在4°C下以3000rpm离心15min,将上层血清转移至洁净Ep管,再在4°C下以15,OOOrpm离心30min以去除细胞碎片,吸取上层血清于洁净离心管,_80°C冻存备用;按照mirVana PARIS试剂盒使用说明书从血清中提取总RNA,使用kandrop测定RNA提取液的A260吸光度值和A260/A280的比值,以评估其浓度;变性琼脂糖凝胶电泳检测RNA的完整性,然后参照DNase I使用说明书,于37°C对RNA提取液消化30min,于-80°C保存备用;(2)Hsa-miR-16cDNA 的合成反转录体系包括:反转录酶0. 5 μ 1、反转录缓冲液0. 75 μ 1、RNA酶抑制剂0. 095 μ 1, hsa-miR-16莲环结构 A kit for the prediction method of the mortality of patients with sepsis as claimed in any one of the use, comprising the steps of: (1) Case of serum samples RNA extraction and collection tube coagulant to a procoagulant, clinical in a conventional Ficoll serum samples with gel separator for collection tube, blood samples were drawn and stay within normal human blood sample M ICU patients septic hours, centrifuged at 3000rpm 15min, the upper layer of serum was transferred to a clean tube Ep at 4 ° C, then at 4 ° C 15, OOOrpm 30min to remove cell debris by centrifugation, the upper layer to absorb the serum in a clean centrifuge tube, _80 ° C cryopreservation standby; kandrop measurement RNA total RNA was extracted from serum according to the mirVana PARIS kit manual, using extract and A260 absorbance ratio A260 / A280, and to assess its concentration; integrity denaturing agarose gel electrophoresis of RNA and DNase I reference manual, at 37 ° C to extract RNA digestion 30min, to - 80 ° C were stored; (2) Hsa-miR-16cDNA synthesis reverse transcription system comprising: a reverse transcriptase 0. 5 μ 1, reverse transcription buffer 0. 75 μ 1, RNA inhibitors 0.095 μ 1, hsa-miR-16 lotus ring structure 物1. 5 μ 1、去离子水2. 08 μ 1、dNTP mix 0. 075 μ 1、及消化后RNA 样品2. 5μ1,共计7. 5μ1 ;将以上成分充分混勻、离心后进行反转录,反转录参数设置为: 160C 30min,42°C 30min,再以85°C 5min灭活反转录酶,合成的cDNA置_20°C冻存备用;(3) hsa-miR-16实时荧光定量PCR的检测扩增系统包括:含Taq酶的miRNA表达定量检测混合液IOul、hsa-miR-16扩增引物与hsa-miR-16荧光标记探针混合液Iul ;向其中加入1. 33ul的反转录产物和7. 67ul的去离子水;利用ABI7300系统进行miRNA定量检测,循环参数设定为95°C IOmin活化Taq酶, 然后95°C 15sec,60°C lmin,共40个循环;选用TOsnRNA为内参,TOsnRNA的检测使用与hsa-miR-16相同的反转录和荧光定量PCR反应体系以及TOsnRNA的引物与探针混合液;(4)hsa-miR-16脓毒症患者中表达情况分析通过与内参TOsnRNA和正常人的血清含量标准化后计算的2_δδ"水平来计算血清中hsa-m Was 1. 5 μ 1, deionized water 2. 08 μ 1, dNTP mix 0. 075 μ 1, and digested RNA sample 2. 5μ1, totaling 7. 5μ1; The above ingredients are thoroughly mixed, after centrifugation inverted record, reverse transcription parameter setting: 160C 30min, 42 ° C 30min, and then at 85 ° C 5min to inactivate reverse transcriptase, the cDNA synthesis frozen spare opposing _20 ° C; (3) hsa-miR-16 real-time quantitative PCR amplification detection system comprising: containing Taq enzyme mixture quantitative detection of miRNA expression IOul, hsa-miR-16 and the amplification primer hsa-miR-16 fluorescent probe mixture IUL; 1 was added thereto. 33ul of the reverse transcription product and 7. 67ul of deionized water; using ABI7300 system for quantitative detection of miRNA, cycle parameter is set to 95 ° C IOmin activation of Taq polymerase, and 95 ° C 15sec, 60 ° C lmin, a total of 40 cycle; TOsnRNA chosen as internal control, TOsnRNA detected using the same hsa-miR-16 reverse transcription and quantitative PCR reaction system and the primers and probes TOsnRNA mixture; (4) hsa-miR-16 in patients with sepsis analysis of the expression of hsa-m is calculated by serum 2_δδ "level and the internal reference serum levels after normalization and normal calculation TOsnRNA iR-16的表达水平,凡是表达水平< 1的患者在28内病死几率大,而表达水平> 1的患者28天病死几率小。 IR-16 expression level, the expression level of all <1 in 28 patients died great chance, while the expression level of> 1 patient died 28 days little chance.
  5. 5.如权利要求4所述的方法,其特在在于使用kandrop测定的RNA提取液的A260吸光度值和A260/A280的比值为1. 9-2. 1 5. The method according to claim 4, which is special in that the A260 absorbance value was used for RNA extraction and measurement kandrop ratio A260 / A280 of 1. 9-2. 1
  6. 6.如权利要求1所述试剂盒用于预测脓毒症患者病死率的用途 6. The kit of claim 1 for use predict mortality in patients with sepsis
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CN103146833A (en) * 2013-03-22 2013-06-12 何蕾 Fluorogenic quantitative PCR (polymerase chain reaction) kit for detecting mice SEPS1 (selenoprotein) gene

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