Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR

Sezigen, Sermet; KENAR, LEVENT; OKTEM, Hüseyin; EYİSON, Koray; ORTATATLI, Mesut

doi:10.1515/tjb-2018-0292

Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR

Levent KENAR, (University of Health Sciences, Department of Medical CBRN Defense, Ankara, Turkey)

Mesut ORTATATLI, (Gulhane Training and Research Hospital, Department of Infectious Diseases and Clinical Microbiology, Ankara, Turkey)

Sermet SEZİGEN, (University of Health Sciences, Department of Medical CBRN Defense, Ankara, Turkey)

Koray EYİSON, (University of Health Sciences, Department of Medical CBRN Defense, Ankara, Turkey)

Hüseyin OKTEM (Middle East Technical University, Department of Biological Sciences, Ankara, Turkey)

Türk Biyokimya Dergisi

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Yıl: 2019 Cilt: 44 Sayı: 2 Sayfa Aralığı: 147 - 152 Metin Dili: İngilizce DOI: 10.1515/tjb-2018-0292 İndeks Tarihi: 06-06-2020

Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR

Öz:

Background: Due to recent increase in mailings of anthraxspores, the detection of bioweapons has gained a greatdeal of interest. This study aimed to investigate the yieldand purity of DNA obtained from spores and vegetativeforms of Bacillus anthracis for detection by conventional(wet) and dry (lyophilized) PCR methods.Materials and methods: Biosamples from stock solution were reconstituted to a concentration 108 cfu/mL followed by the spectrophotometric measurement of theyield and purity of acquired DNA. Twelve wet and 12 dryPCR studies of four various DNA dilution samples wereperformed for each three target gene (cap, pag, sap) ofB. anthracis.Results: Significant differences for both DNA yields andpurity were found between liquid-agar and liquid-sporesamples. No significant difference was observed betweenwet and dry PCR in concentration of 2.5 ng/μL DNA for allgene regions. PCR results of sap gene region with DNAconcentrations of 1.5 ng/μL and 0.9 ng/μL was foundstatistically significant in favor of conventional wet PCRmethod (p = 0.047 and p ≤ 0.001, respectively).Conclusion: It is concluded that B. anthracis cultivatedin liquid medium is more usable than vegetative or sporeform obtained on plate agar for proper DNA extraction.

Anahtar Kelime:

Bacillus anthracis’in moleküler tespiti: DNA ekstraksiyonu ve yeni bir kuru PCR yönteminin etkinliğinin değerlendirilmesi

Öz:

Amaç: Son zamanlarda antraks sporlarının postalayla gönderilmesindeki artış nedeniyle, biyolojik silahların tespiti büyük önem kazanmıştır. Bu çalışma, Bacillus anthracis’in konvansiyonel (ıslak) ve kuru (liyofilize) PCR yöntemleri ile saptanmasında spor ve vejetatif formlardan elde edilen DNA’nın miktar ve saflığını araştırmayı amaçlamıştır. Gereç ve Yöntem: Stok çözeltiden elde edilen numuneler 108cfu/mL konsantrasyona getirildi, ardından elde edilen DNA’nın miktar ve saflığı spektrofotometrik yöntemle ölçüldü. Dört farklı DNA dilüsyonunda B. anthracis’in üç hedef gen bölgesine (cap, pag, sap) yönelik 12 ıslak ve 12 kuru PCR çalışması gerçekleştirildi. Bulgular: Sıvı-agar ve sıvı-spor örnekleri arasında hem DNA miktarı hem de saflığı açısından istatistiksel önemli fark bulundu. Tüm gen bölgeleri için 2.5 ng/μL DNA konsantrasyonunda ıslak ve kuru PCR arasında anlamlı bir fark gözlenmedi. Sap gen bölgesine yönelik uygulanan PCR’da, 1.5 ng/μL ve 0.9 ng/μL DNA konsantrasyonlarında konvansiyonel ıslak PCR yöntemi lehine istatistiksel anlamlı fark belirlendi (sırasıyla, p = 0.047 ve p ≤ 0.001). Sonuç: Uygun DNA ekstraksiyonu açısından sıvı ortamda üretilen B. anthracis’in agarda üretilen vejetatif veya spor formundan daha uygun olduğu sonucuna varılmıştır.

Anahtar Kelime:

Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık

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APA	KENAR L, ORTATATLI M, Sezigen S, EYİSON K, OKTEM H (2019). Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. , 147 - 152. 10.1515/tjb-2018-0292
Chicago	KENAR LEVENT,ORTATATLI Mesut,Sezigen Sermet,EYİSON Koray,OKTEM Hüseyin Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. (2019): 147 - 152. 10.1515/tjb-2018-0292
MLA	KENAR LEVENT,ORTATATLI Mesut,Sezigen Sermet,EYİSON Koray,OKTEM Hüseyin Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. , 2019, ss.147 - 152. 10.1515/tjb-2018-0292
AMA	KENAR L,ORTATATLI M,Sezigen S,EYİSON K,OKTEM H Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. . 2019; 147 - 152. 10.1515/tjb-2018-0292
Vancouver	KENAR L,ORTATATLI M,Sezigen S,EYİSON K,OKTEM H Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. . 2019; 147 - 152. 10.1515/tjb-2018-0292
IEEE	KENAR L,ORTATATLI M,Sezigen S,EYİSON K,OKTEM H "Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR." , ss.147 - 152, 2019. 10.1515/tjb-2018-0292
ISNAD	KENAR, LEVENT vd. "Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR". (2019), 147-152. https://doi.org/10.1515/tjb-2018-0292

APA	KENAR L, ORTATATLI M, Sezigen S, EYİSON K, OKTEM H (2019). Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. Türk Biyokimya Dergisi, 44(2), 147 - 152. 10.1515/tjb-2018-0292
Chicago	KENAR LEVENT,ORTATATLI Mesut,Sezigen Sermet,EYİSON Koray,OKTEM Hüseyin Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. Türk Biyokimya Dergisi 44, no.2 (2019): 147 - 152. 10.1515/tjb-2018-0292
MLA	KENAR LEVENT,ORTATATLI Mesut,Sezigen Sermet,EYİSON Koray,OKTEM Hüseyin Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. Türk Biyokimya Dergisi, vol.44, no.2, 2019, ss.147 - 152. 10.1515/tjb-2018-0292
AMA	KENAR L,ORTATATLI M,Sezigen S,EYİSON K,OKTEM H Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. Türk Biyokimya Dergisi. 2019; 44(2): 147 - 152. 10.1515/tjb-2018-0292
Vancouver	KENAR L,ORTATATLI M,Sezigen S,EYİSON K,OKTEM H Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR. Türk Biyokimya Dergisi. 2019; 44(2): 147 - 152. 10.1515/tjb-2018-0292
IEEE	KENAR L,ORTATATLI M,Sezigen S,EYİSON K,OKTEM H "Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR." Türk Biyokimya Dergisi, 44, ss.147 - 152, 2019. 10.1515/tjb-2018-0292
ISNAD	KENAR, LEVENT vd. "Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR". Türk Biyokimya Dergisi 44/2 (2019), 147-152. https://doi.org/10.1515/tjb-2018-0292