 Corresponding author: Rana H. Raheema Department of Medical Microbiology, Faculty of Medicine, University of Wasit, Iraq. Copyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Design of primers for evaluation of some gram-negative bacteria isolated from intensive care unit patients in Wasit province, Iraq Rana Essa Muslem and Rana H. Raheema * Department of Medical Microbiology, Faculty of Medicine, University of Wasit, Iraq. World Journal of Advanced Research and Reviews, 2022, 16(02), 825–832 Publication history: Received on 25 September 2022; revised on 11 November 2022; accepted on 14 November 2022 Article DOI: https://doi.org/10.30574/wjarr.2022.16.2.1133 Abstract Intensive care unit (ICU) patients frequently have consequences from healthcare-associated infections (HAIs), which include bacteremia, pneumonia, urinary tract, skin, or soft tissue infections. . In this study, a total of 100 clinical specimens (urine, sputum and pus) were collected from patients admitted in the ICU. Results showed eighty two were positive growth culture and admitted to the intensive care unit distributed 1 (1.2%) Klebsiella oxytoca, 5(6.25%)Proteus mirabilis, 2 (2.4%) Acinetobacter baumannii , 1 (1.2%) Serratia marcescens and 1 (1.2%) Burkholderia cepacia group bacteria . The results of this study showed that the diagnostic test for 16S rRNA bacteria (Proteus mirabilis), which numbered 5 (100%), bacteria (Acinetobacter baumannii), which numbered 2 (100%), bacteria (Burkholderia cepacia ), which numbered 1 (100%), bacteria (Serratia marcescens ), which numbered 1 (100%), and Bacteria (klebsiella oxytoca ), which numbered 1 (100%) after completing the phenotypic and biochemical diagnosis diagnostic test (16S rRNA). Keywords: 16S rRNA; Acinetobacter baumannii; Proteus mirabilis; Serratia marcescens; Klebsiella oxytoca; Burkholderia cepacia complex; Intensive care unit 1. Introduction Intensive care unit (ICU) patients frequently have consequences from healthcare-associated infections (HAIs), which include bacteremia, pneumonia, urinary tract, skin, or soft tissue infections [1]. The ICU personnel and doctors may act as conduits for the transfer of germs from other inpatient units to ICUs [2]. Therefore, HCWs and ICU staff must maintain the highest levels of hand cleanliness. The ICU environment's contamination also has a significant impact on how patients and HCWs pick up nosocomial bacteria [3]. The gram-negative bacteria that recorded in the intensive care unit are Enterobacteriaceae, including Acinetobacter baumannii, Proteus mirabilis, Serratia marcescens, klebsiella oxytoca, and Burkholderia cepacia complex [4]. Determining the spread of gram negative bacteria from intensive care unit patients in Kut City, Wasit Province, Iraq and characterizing it at the molecular level through analyses of 16S rRNA genes. 2. Material and methods A cross-sectional study was done in the intensive care unit (ICU) of the Alzahraa , Alkarama hospitals from the 3 rd October 2021 to 20 th February 2022 . A total of 100 clinical samples, including: urine, sputum and pus culture media such as mannitol salt agar, MacConkey agar, blood agar, and chocolate agar. The growth showed different bacterial colonies whose morphological and biochemical characteristics were tested. Then DNA was extracted; purity and concentration were confirmed with Nanodrop. The purity of gram negative bacteria (1.8-2), and the concentration was between 50-360 ng/µl. Primers were mentioned as the procedure by [5].