title: The potential role of incorporating real-time PCR and DNA sequencing for amplification and detection of 16S rRNA gene signatures in neonatal sepsis.
abstract: OBJECTIVE
  This study aimed to explore whether 16S rRNA gene amplification by real time PCR and sequencing could serve as genetic-based methods in rapid and accurate diagnosis of neonatal sepsis.
  
  
  METHODS
  This case control study was conducted on 40 neonates suffering from sepsis like manifestations recruited from the neonatal intensive care unit of Menoufia university hospital over a period of 6 months. Their blood samples were used for paired analysis of bacterial growth using BACTEC 9050 instrument and real time PCR assay with subsequent DNA sequencing for bacterial species identification.
  
  
  RESULTS
  The detection rate of culture proven sepsis was 70%. By using real time 16S r RNA PCR amplification method, the detection of bacteria was improved to 80%. Real time PCR revealed sensitivity, specificity, positive predictive value and negative predictive value of [100%, 66.7%, 87.5% and 100%] respectively. Compared to culture, the 16S rRNA real time PCR demonstrated a high negative value for ruling out neonatal sepsis. There was significant statistical difference between the PCR positive and negative cases as regards the hematological sepsis score. The results demonstrated the ability of DNA sequencing to recognize 4 pathogens which were negative by blood culture. The time consumed to detect sepsis using blood culture was up to 5 days while it took up to 16 h only by PCR and sequencing methods.
  
  
  CONCLUSIONS
  16S rRNA gene amplification by real time PCR and sequence analysis could be served as ideal and reliable genetic-based methods to diagnose and rule out sepsis with provision of additional data that cannot be obtained by routine laboratory tests with a shorter turnaround time than those with culture-based protocols.
authors.affiliation: a Department of Pediatrics , Faculty of Medicine, Menoufia University , Shebin el kom , Menoufia , Egypt and.
authors.forename: Dina A
authors.initials: DA
authors.lastname: Midan
authors.affiliation: a Department of Pediatrics , Faculty of Medicine, Menoufia University , Shebin el kom , Menoufia , Egypt and.
authors.forename: Wafaa Moustafa M
authors.initials: WMM
authors.lastname: Abo El Fotoh
authors.affiliation: b Department of Clinical Pathology , Faculty of Medicine, Menoufia University , Shebin el kom , Menoufia , Egypt.
authors.forename: Abeer H
authors.initials: AH
authors.lastname: El Shalakany
mesh_terms: D016022:Case-Control Studies; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D000071074:Neonatal Sepsis / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology; D012336:RNA, Ribosomal, 16S / Q000097:blood* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D013210:Staphylococcus / Q000235:genetics / Q000302:isolation & purification
chemical_list: D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA; Neonates; blood culture; real time PCR; sepsis; sequencing