应用芯片式数字PCR检测人血清中登革病毒载量
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摘要
目的建立芯片式数字PCR绝对定量技术,用于登革病例血清中登革病毒载量绝对定量并分析载量变化情况。方法选取发病1～7d登革实验室确诊病例血清样本,提取病毒RNA并逆转录为cDNA,取3μL或4. 5μL cDNA,配制15μL反应体系,采用Quant Studio 3D Digital PCR Chip Loader仪器制作芯片,将芯片置于Dual Flat Block Gene Amp PCR System 9700运行PCR,最后在Quant Studio~(TM)3D Digital PCR System中读取结果并应用Quant Studio~(TM)3D Analysis Suite~(TM) Software进行分析,根据实验稀释倍数计算血清中登革病毒载量。结果芯片制作良好,有效孔数高,检测结果重复性较好,示病例发病第1～7d血清中的登革病毒载量分别是10~(7. 646),10~(7. 544),10~(7. 298),10~(6. 990),10~(5. 823),10~(4. 732),10~(4. 221)copies/m L。结论应用芯片式数字PCR检测血清中登革病毒载量的方法可行,可应用于登革病毒载量的绝对定量。登革病毒载量随着病程的发展而降低。
Objective To establish a chip digital PCR quantitative technique for absolute quantification of serum dengue virus load and to analyze the changes of dengue virus load in the serum of patients. Methods The serum samples of laboratory confirmed dengue cases were extracted. Virus RNA was extracted and reverse-transcribed into cDNA. Three or 4. 5 μL cDNA was used to prepare 15 μL reaction system. The chip was made by Quant Studio 3D Digital PCR Chip Loader instrument,and then was placed in Dual Flat Block Gene Amp PCR System 9700 to run PCR. The results were read in Quant Studio TM 3D Digital PCR System,and the dengue virus load in human serum was calculated by using Quant Studio 3D Analysis Suite Software. Results The digital PCR chip wasmade well,the number of effective holes was high,and the test results were reproducible. The results showed that the dengue virus load in serum on the 1st through 7th day of onset was 10~(7. 646),10~(7. 544),10~(7. 298),10~(6. 990),10~(5. 823),10~(4. 732),and 10~(4. 221) copies/m L,respectively.Conclusion The method of using chip digital PCR to detect the dengue virus load in human serum was feasible and can be applied to the absolute quantification of the dengue virus load. It was found that the dengue virus load decreased with the development of the course of disease.
Objective To establish a chip digital PCR quantitative technique for absolute quantification of serum dengue virus load and to analyze the changes of dengue virus load in the serum of patients. Methods The serum samples of laboratory confirmed dengue cases were extracted. Virus RNA was extracted and reverse-transcribed into cDNA. Three or 4. 5 μL cDNA was used to prepare 15 μL reaction system. The chip was made by Quant Studio 3D Digital PCR Chip Loader instrument,and then was placed in Dual Flat Block Gene Amp PCR System 9700 to run PCR. The results were read in Quant Studio TM 3D Digital PCR System,and the dengue virus load in human serum was calculated by using Quant Studio 3D Analysis Suite Software. Results The digital PCR chip wasmade well,the number of effective holes was high,and the test results were reproducible. The results showed that the dengue virus load in serum on the 1st through 7th day of onset was 10~(7. 646),10~(7. 544),10~(7. 298),10~(6. 990),10~(5. 823),10~(4. 732),and 10~(4. 221) copies/m L,respectively.Conclusion The method of using chip digital PCR to detect the dengue virus load in human serum was feasible and can be applied to the absolute quantification of the dengue virus load. It was found that the dengue virus load decreased with the development of the course of disease.
引文
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[11] SAHA P,GEISSMANN F. Toward a functional characterization of blood monocytes[J]. Immunol Cell Biol,2011,89(1):2-4.
[12]王茜茜,洪文昕,邱爽,等.登革热发生中单核细胞CD14~+CD16~+亚群的增殖变化及病情与病毒载量的关系[J].中山大学学报(医学科学版),2016,37(3):337-342.
[13] VAUGHN D W,GREEN S,KALAYANAROOJ S,et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity[J]. The Journal of infectious diseases,2000(181):2-9.
[14]梅建华,雷永良,陈秀英,等.基于芯片式数字PCR技术的乙型肝炎病毒绝对定量检测方法的建立及应用[J].中国卫生检验杂志,2018(8):925-928.
[15] WACKER M J. Analysis of one-step and two-step real-time rtPCR using superscript III[J]. J Biomol Tech,2005,16(3):266-271.
[2]潘欢欢,李锋平. 2007—2016年福建泉州市登革热流行特征分析[J].公共卫生与预防医学,2017,8(5):30-32.
[3]周航,李昱,牟笛,等.中国2013年登革热流行病学分析[J].公共卫生与预防医学,2015,26(2):12-15.
[4] SANDERS R,HUGGETT J F,BUSHELL C A,et al. Evaluation of digital PCR for absolute DNA quantification[J]. Anal Chem,2011(83):6474-6484.
[5] PERSSON S,ERIKSSON R,LOWTHER J,et al. Comparison between RT droplet digital PCR and RT real-time PCR for quantification of noroviruses in oysters[J]. International journal of food microbiology,2018(284):73-83.
[6] ABACHIN E,CONVERS S,FALQUE S,et al. Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid[J]. Biologicals:journal of the International Association of Biological Standardization,2018(52):49-54.
[7] TANG H,CAI Q,LI H,et al. Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA[J].Biosci Biotechnol Biochem,2016:1-6.
[8]冯兆民.基于微滴式数字PCR技术的甲型流感病毒绝对定量研究[D].北京:中国疾病预防控制中心,2017.
[9]赵令斋,高秀洁,庾蕾,等.登革病毒载量和Ig M抗体水平与疾病严重程度的关系[J].中华微生物学和免疫学杂志,2016(4):36-44.
[10]赵令斋.登革病毒基因变异、病毒载量及机体固有免疫与登革热严重程度的相关性研究[D].广州:广州医科大学,2017.
[11] SAHA P,GEISSMANN F. Toward a functional characterization of blood monocytes[J]. Immunol Cell Biol,2011,89(1):2-4.
[12]王茜茜,洪文昕,邱爽,等.登革热发生中单核细胞CD14~+CD16~+亚群的增殖变化及病情与病毒载量的关系[J].中山大学学报(医学科学版),2016,37(3):337-342.
[13] VAUGHN D W,GREEN S,KALAYANAROOJ S,et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity[J]. The Journal of infectious diseases,2000(181):2-9.
[14]梅建华,雷永良,陈秀英,等.基于芯片式数字PCR技术的乙型肝炎病毒绝对定量检测方法的建立及应用[J].中国卫生检验杂志,2018(8):925-928.
[15] WACKER M J. Analysis of one-step and two-step real-time rtPCR using superscript III[J]. J Biomol Tech,2005,16(3):266-271.