Position: Home > Articles > Detection of Gluconacetobacter in Yogurt by Helicase-Dependent Isothermal DNA Amplification
Journal of Dairy Science and Technology
2017,40
(2)
30-33
酸乳中葡糖醋杆菌赖解旋酶恒温基因扩增检测方法的建立
作 者：
唐心怡;刘波;张涛;李永艳;张翠侠;王赞;章晶晶;周巍
单 位：
河北省食品检验研究院;北京市第十二中学
关键词：
赖解旋酶恒温基因扩增法;酸乳;葡糖醋杆菌;检测
摘 要：
建立酸乳中葡糖醋杆菌的赖解旋酶恒温基因扩增(helicase-dependent isothermal DNA amplification,HDA)检测方法。根据葡糖醋杆菌16S基因序列(基因号为HQ677466.1)设计特异性引物,并优化反应体系中UvrD解旋酶和T4 gp32的浓度。通过HDA法直接检测酸乳中的葡糖醋杆菌,并确定其检出限,在建立的HDA体系中对酸乳中多种菌株进行扩增和电泳检测,验证方法的特异性。结果表明:用HDA法检测酸乳中葡糖醋杆菌,得到了与设计序列长度(101 bp)一致的基因片段,检出限为102 CFU/g,反应体系中UvrD解旋酶和T4 gp32的适宜添加量分别为0.1μg和5.0μg。该方法用于检测酸乳中葡糖醋杆菌的灵敏度高、耗时短。
译 名：
Detection of Gluconacetobacter in Yogurt by Helicase-Dependent Isothermal DNA Amplification
作 者：
TANG Xinyi;LIU Bo;ZHANG Tao;LI Yongyan;ZHANG Cuixia;WANG Zan;ZHANG Jingjing;ZHOU Wei;Beijing No.12 High School;Hebei Key Laboratory of Food Safety, Hebei Food Inspection and Research Institute;
关键词：
helicase-dependent isothermal DNA amplification(HDA);;yogurt;;Gluconacetobacter;;detection
摘 要：
A helicase-dependent isothermal DNA amplification(HDA) assay was developed for the rapid and accurate detection of Gluconacetobacter in yogurt. A pair of specific oligonucleotide primers according to the 16 S ribosomal RNA gene(Gen Bank accession number: HQ677466.1) of Gluconacetobacter was designed, and the optimal concentration of UvrD helicase and T4 gp32 in the reaction system were determined. The HAD method enabled direct detection of Gluconacetobacter in yogurt. The specificity of the method was tested by amplification and electrophoresis of various strains in yoghurt using the HAD system. The amplification product by HDA was the same as the designed gene fragment(101 bp). The sensitivity of HDA was 10~2 CFU/g. The optimal reaction system was established by the use of 0.1 μg of UvrD helicase and 5.0 μg of T4 gp32. In conclusion, the method was sensitive and time saving.
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