Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay.
DNA extraction efficiency affects the success of PCR-based method applications. The Punch-it™ NA-Sample kit for extracting DNA by using paper chromatography is technically easy to use and requires just two reagents and only 10min to complete. The Punch-it™ NA-Sample kit could be offered as a rapid, accurate, and convenient method for extracting bacterial and fungal DNA from blood culture bottles. We compared the efficiencies of the commercial kit (Punch-it™ NA-Sample kit) and an in-house conventional boiling method with Chelex-100 resin for DNA extraction from blood culture bottles. The efficiency of the two DNA extraction methods was assessed by PCR-reverse blot hybridization assay (PCR-REBA, REBA Sepsis-ID) for detecting Gram positive (GP) bacteria, Gram negative (GN) bacteria, and Candida species with 196 positive and 200 negative blood culture bottles. The detection limits of the two DNA extraction methods were 10(3)CFU/mL for GP bacteria, 10(3)CFU/mL for GN bacteria, and 10(4)CFU/mL for Candida. The sensitivity and specificity of the Punch-it™ NA-Sample kit by REBA Sepsis-ID were 95.4% (187/196) and 100% (200/200), respectively. The overall agreement of the two DNA extraction methods was 98.9% (392/396). Three of four samples showing discrepant results between the two extraction methods were more accurately matched up with the Punch-it™ NA-Sample kit based on conventional culture methods. The results indicated that the Punch-it™ NA-Sample kit extracted bacterial and fungal DNA in blood culture bottles and allowed extracted DNA to be used in molecular assay.
Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju-si, Gangwon-do 26493, Republic of Korea.
Jungho
J
Kim
M&D, Inc., Wonju Eco Environmental Technology Center, Wonju-si, Gangwon-do 26493, Republic of Korea.
Hye-Young
HY
Wang
Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju-si, Gangwon-do 26493, Republic of Korea.
Seoyong
S
Kim
Department of Laboratory Medicine, Yonsei University, Wonju College of Medicine, 20 Ilsan-ro, Wonju-si, Gangwon-do 26426, Republic of Korea.
Soon Deok
SD
Park
Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju-si, Gangwon-do 26493, Republic of Korea.
Kwangmin
K
Yu
Department of Internal Medicine, Yonsei University, Wonju College of Medicine, 20 Ilsan-ro, Wonju-si, Gangwon-do 26426, Republic of Korea.
Hyo Youl
HY
Kim
Department of Laboratory Medicine, Yonsei University, Wonju College of Medicine, 20 Ilsan-ro, Wonju-si, Gangwon-do 26426, Republic of Korea. Electronic address: u931018@yonsei.ac.kr.
Young
Y
Uh
Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju-si, Gangwon-do 26493, Republic of Korea. Electronic address: hyelee@yonsei.ac.kr.
Hyeyoung
H
Lee
D000071997:Blood Culture; D002175:Candida / Q000302:isolation & purification; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000302:isolation & purification*; D006090:Gram-Negative Bacteria / Q000302:isolation & purification; D006094:Gram-Positive Bacteria / Q000302:isolation & purification; D057230:Limit of Detection; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000175:diagnosis / Q000382:microbiology; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D004271:DNA, Fungal; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic
Bacteria; Blood culture; DNA extraction; Hybridization; Molecular diagnosis