Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection

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• 作者：Johan H. Melendez^a ; Tonya M. Santaus^a ; Gregory Brinsley^a ; Daniel Kiang^a ; Buddha Mali^a ; Justin Hardick^b ; Charlotte A. Gaydos^b ; Chris D. Geddes^a ; ^{geddes@umbc.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Neisseria gonorrhoeae ; Gonorrhea ; Microwaves ; DNA extraction ; DNA fragmentation ; Microwave-accelerated metal-enhanced fluorescence
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：1 October 2016
• 年：2016
• 卷：510
• 期：Complete
• 页码：33-40
• 全文大小：1452 K

文摘

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections.