Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay

Jungho Kim, Hye young Wang, Seoyong Kim, Soon Deok Park, Kwangmin Yu, Hyo Youl Kim, Young Uh, Hyeyoung Lee

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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 10 min 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.

Original languageEnglish
Pages (from-to)24-30
Number of pages7
JournalJournal of Microbiological Methods
Volume128
DOIs
Publication statusPublished - 2016 Sep 1

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Polymerase Chain Reaction
DNA
Fungal DNA
Bacterial DNA
Gram-Positive Bacteria
Gram-Negative Bacteria
Candida
Sepsis
Paper Chromatography
Blood Culture
Limit of Detection
Sensitivity and Specificity

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

Cite this

Kim, Jungho ; Wang, Hye young ; Kim, Seoyong ; Park, Soon Deok ; Yu, Kwangmin ; Kim, Hyo Youl ; Uh, Young ; Lee, Hyeyoung. / Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay. In: Journal of Microbiological Methods. 2016 ; Vol. 128. pp. 24-30.
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abstract = "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 10 min 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.",
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Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay. / Kim, Jungho; Wang, Hye young; Kim, Seoyong; Park, Soon Deok; Yu, Kwangmin; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung.

In: Journal of Microbiological Methods, Vol. 128, 01.09.2016, p. 24-30.

Research output: Contribution to journalArticle

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