“新筛”干血斑在耳聋基因检测中的应用

doi:10.3969/j.issn.1673-8640.2020.03.016

摘要/Abstract

摘要：

目的将新生儿遗传代谢病筛查（简称“新筛”）后剩下的干血斑继续用于耳聋基因提取和飞行质谱检测,从而减少耳聋基因检测过程中的取样频次并缩短检测周期。方法在碱水煮沸法提取耳聋基因的基础上优化碱水的pH值和用量,使“新筛”干血斑DNA提取浓度达到新鲜血斑水平。结果用pH值为9、10、11、12的碱水从干血斑中提取DNA时,DNA的浓度与碱水pH值呈正相关（r=0.906,P=0.094）,20个耳聋基因位点均能被正常检出;当pH值达到12时,质谱峰图信号响应值减小,检测质量下降。用60、70、80、90 μL pH值为11的碱水从“新筛”干血斑中提取DNA时,DNA浓度与碱水体积呈负相关（r=-0.988,P=0.012）;用90 μL pH值为11的碱水时,有1例样本547G>A位点基因被漏检,用80 μL pH值为11的碱水时,DNA提取浓度和新鲜血斑最接近（t=0.035,P=0.974）,同时质谱峰图良好。结论用80 μL pH值为11的煮沸碱水从“新筛”干血斑中提取DNA,可获得较好的耳聋质谱检测结果,为搭建“新筛”和耳聋自动化检测平台提供一定的技术参考。

关键词: 干血斑, 新生儿遗传代谢病筛查, 耳聋, 提取

Abstract:

Objective To reduce sampling frequency and shorten deafness gene detection period in detecting deafness gene by time-of-flight mass spectrometry with discarded dry blood spot generated from newborn screening. Methods The DNA extracting method of boiling discarded dry blood spot with alkaline water was optimized by adjusting the pH value and the dosage of alkaline water. Results When extracting DNA from discarded dry blood spot by alkaline water with the pH values of 9,10,11 and 12,all the 20 deafness gene were accurately detected,and the concentration of DNA was positively correlated with the pH value of alkaline water（r=0.906,P=0.094）,however,the DNA extracted from alkaline water with pH12 was difficult to be detected,as the peak value got sharply decreased. When extracting DNA from discarded dry blood spot by alkaline water with the pH value of 11 and the volumes of 60,70,80 and 90 μL,the concentration of DNA was negatively correlated with the volume of alkaline water（r=-0.988,P=0.012）,and the genetic locus of 547G>A in one samples extracted from 90 μL alkaline water with the pH value of 11 was failed to be detected,while the concentration of DNA extracted from discarded dry blood spot with 80 μL alkaline water with the pH value of 11 was close to those extracted from fresh dry blood spot（t=0.035,P=0.974）,and all the 20 genetic locuses related to deafness were accurately detected as well. Conclusions Good detection results could be obtained when extracting DNA from discarded dry blood spot through 80 μL alkaline water with the pH value of 11,and this study provides a reference on the construction of automatic detection platform for both newborn screening of genetic metabolic disease and deafness gene detection.

Key words: Dry blood spot, Newborn screening for genetic and metabolic diseases, Deafness, Extraction

中图分类号:

R446.1

高鹏, 覃诗琪, 唐美芳. “新筛”干血斑在耳聋基因检测中的应用[J]. 检验医学, 2020, 35(3): 264-269.

GAO Peng, QIN Shiqi, TANG Meifang. Application of dry blood spot from newborn screening in deafness gene detection[J]. Laboratory Medicine, 2020, 35(3): 264-269.

图/表 6

参考文献 12

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血斑类型	核酸提取浓度/（ng/mL）	未检出位点/个
干血斑
S1	0.15	0
S2	0.16	1
S3	0.11	1
S4	0.14	1
S5	0.15	0
S6	0.12	1
新鲜血斑
S1	0.11	0
S2	0.38	0
S3	0.28	0
S4	0.34	0
S5	0.24	0
S6	0.25	0

血斑类型	核酸提取浓度/（ng/mL）	未检出位点/个
干血斑
S1	0.15	0
S2	0.16	1
S3	0.11	1
S4	0.14	1
S5	0.15	0
S6	0.12	1
新鲜血斑
S1	0.11	0
S2	0.38	0
S3	0.28	0
S4	0.34	0
S5	0.24	0
S6	0.25	0

位点	pH值9		pH值10		pH值11		pH值12		对照
位点	洗	不洗	洗	不洗	洗	不洗	水洗	不洗	洗
1174A>T	A	A	A	A	A	A	A	A	A
1226G>A	A	A	A	A	A	A	C	B	A
1229C>T	A	A	A	A	A	A	A	A	A
1494C>T	A	A	A	A	A	A	A	A	A
1555A>G	A	A	A	A	A	A	B	A	A
167delT	B	B	B	B	B	B	A	B	B
176_191del16	A	A	A	A	A	A	A	B	A
1975G>C	A	A	A	A	A	A	A	A	B
2027T>A	A	A	A	A	A	A	A	A	A
2162C>T	A	A	A	A	A	A	B	B	A
2168A>G	A	A	A	A	A	A	A	A	A
235delC	A	A	A	A	A	A	A	A	A
281C>T	A	A	A	A	A	A	A	B	A
299_300delAT	A	A	A	A	A	A	A	A	A
35delG	A	A	A	A	A	A	A	A	A
538C>T	A	A	A	A	A	A	A	A	A
547G>A	A	A	A	A	A	A	A	A	A
589G>A	A	A	A	A	A	A	A	A	A
IVS15+5G>A	A	A	A	A	A	A	A	A	A
IVS7-2A>G	A	A	A	A	A	A	A	A	A

位点	pH值9		pH值10		pH值11		pH值12		对照
位点	洗	不洗	洗	不洗	洗	不洗	水洗	不洗	洗
1174A>T	A	A	A	A	A	A	A	A	A
1226G>A	A	A	A	A	A	A	C	B	A
1229C>T	A	A	A	A	A	A	A	A	A
1494C>T	A	A	A	A	A	A	A	A	A
1555A>G	A	A	A	A	A	A	B	A	A
167delT	B	B	B	B	B	B	A	B	B
176_191del16	A	A	A	A	A	A	A	B	A
1975G>C	A	A	A	A	A	A	A	A	B
2027T>A	A	A	A	A	A	A	A	A	A
2162C>T	A	A	A	A	A	A	B	B	A
2168A>G	A	A	A	A	A	A	A	A	A
235delC	A	A	A	A	A	A	A	A	A
281C>T	A	A	A	A	A	A	A	B	A
299_300delAT	A	A	A	A	A	A	A	A	A
35delG	A	A	A	A	A	A	A	A	A
538C>T	A	A	A	A	A	A	A	A	A
547G>A	A	A	A	A	A	A	A	A	A
589G>A	A	A	A	A	A	A	A	A	A
IVS15+5G>A	A	A	A	A	A	A	A	A	A
IVS7-2A>G	A	A	A	A	A	A	A	A	A

项目	干血斑处理情况	DNA提取浓度/（ng/μL）
pH值9	洗	0.15±0.05
	不洗	0.16±0.04
pH值10	洗	0.18±0.04
	不洗	0.17±0.04
pH值11	洗	0.21±0.05
	不洗	0.21±0.05
pH值12	洗	0.23±0.08
	不洗	0.34±0.06^*
对照	洗	0.27±0.09