一种新的化学裂解方法，酒店保洁13825404095可最大程度地释放难裂解细菌中的DNA。
Microbial Pathogenesis
(
IF
3.3
)
Pub Date : 2018-11-12
, DOI:
10.1016/j.micpath.2018.11.008
Olle M de Bruin
1
,
Amy Chiefari
2
,
Danielle Wroblewski
2
,
Christina Egan
2
,
Cassandra D Kelly-Cirino
1
Affiliation
DNA Genotek Inc., 3000 - 500 Palladium Drive, Ottawa, ON, K2V 1C2, Canada.
Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY, 12208, USA.
微生物的分子检测需要从细胞释放DNA。但是，由于某些微生物难以通过化学或酶促方法进行裂解，因此通常采用通过微珠敲打进行的机械裂解来破坏难以裂解的微生物。一种新近开发的化学裂解方法，称为孢子裂解（sporeLYSE），可从难裂解的微生物中释放DNA，而不会发生珠子敲打。将sporeLYSE方法与敲打珠子和碱性/去污剂裂解溶液进行了比较，该溶液可从体外生长的微生物（包括A类生物恐怖剂的替代物）释放DNA。孢子裂解液从耻垢分枝杆菌，费氏弗朗西斯菌，肠炎耶尔森菌，苏云金杆菌，铜绿假单胞菌，卡他莫拉菌和肺炎克雷伯菌中释放了83％至100％的DNA。qPCR结果表明，从革兰氏阳性菌和革兰氏阴性菌中，孢子裂解液提取的DNA等于或不经过磁珠裂解或碱/洗涤剂裂解的DNA量。当使用孢子裂解液分别从耻垢分枝杆菌和结核分枝杆菌掺入的唾液和痰中提取DNA时，qPCR Ct值比通过碱/洗涤剂裂解和加热提取的值低4-8个循环。从艰难梭状芽胞杆菌和肉毒梭菌的孢子中进行LYSE提取的平均Ct值比MagNA Pure DNA提取的平均Ct值低两个周期。我们的结果表明，sporeLYSE是一种易于使用的液体试剂，可以有效地从各种细菌（包括孢子）中释放大量DNA。
A novel chemical lysis method for maximum release of DNA from difficult-to-lyse bacteria.
Molecular detection of microorganisms requires releasing DNA from cells. However, since certain microbial organisms are refractory to lysis by chemical or enzymatic methods, mechanical lysis by bead-beating is typically employed to disrupt difficult-to-lyse microbes. A newly developed chemical lysis method called sporeLYSE enables release of DNA from difficult-to-lyse microbes without bead-beating. The sporeLYSE method was compared to bead-beating and an alkaline/detergent lysis solution for releasing DNA from microbes grown in vitro, including surrogates of Category A bioterrorism agents. sporeLYSE released 83% to 100% of DNA from Mycobacterium smegmatis, Francisella philomiragia, Yersinia enterocolitica, Bacillus thuringiensis, Pseudomonas aeruginosa, Moraxella catarrhalis and Klebsiella pneumoniae. qPCR results indicated that sporeLYSE extracted an equal or greater amount of DNA than either bead-beating or alkaline/detergent lysis from Gram-positive and Gram-negative bacteria. When sporeLYSE was used to extract DNA from saliva and sputum spiked with M. smegmatis and M. tuberculosis, respectively, the qPCR Ct values were 4-8 cycles lower than those for extractions via alkaline/detergent lysis and heat. Mean Ct values for sporesLYSE extractions from spores of Clostridium difficile and C. botulinum were approximately two cycles lower than those of MagNA Pure DNA extractions. Our results suggest that sporeLYSE is an easy-to-use liquid reagent that can efficiently release large amounts of DNA from a variety of bacteria, including spores.
