Forensic molecular study using ITS region as a clue and possible fungal outbreak pre-warning indicator Ali H. Al-Assadi 1 , Mohammad J. Al-Jassani 2* 1 University of Kerbala, College of Pharmacy, Kerbala, Iraq 2 University of Babylon, DNA research center, Hilla, Iraq Abstract : The PCR primers ITS1 and ITS4 are commonly used to gain wide acceptance for work with fungal Internal Transcribe Sequences (ITS) that amplify the highly variable ITS1 and ITS2 regions. ITS region was evaluated for mixed human blood sample differentiation. Some of these fungal pathogens can affect both human and animal and can contaminate the environment, or may establish new enzootic foci. Total of 70 blood samples were collected in the EDTA tube that include (20 healthy (non-symptomatic) human blood samples, 20 sheep blood samples, 20 chicken blood samples and 10 human blood of leukemia patients. Blood culture has showed no results even when Blood agar plates were incubated for a week. PCR result showed that mycosis in human leukemia patient 70% in comparison with 30% in non- symptomatic samples. Positive human blood can be differentiated in mixed samples using this primer pair. Chicken and sheep were found infected by 40% and 20% respectively. Fungal infection detection using ITS region can facilitate determination of geographical area and human involvement in some forensic cases where mixed blood samples are present. Determination of fungal strain aid in putting a cut edge in zoonotic infection outbreak and provide information in possible bio-terrorism attack origin. Key words: Forensic microbiology, ITS region, Fungal infection. Introduction: Microbial forensics is a new branch of the forensics field focuses on the person or people who perpetrated a crime, and on the victims and their related microbiology. Microbial forensic analysis is became very important in the detection of reliable molecular variations among microbial strains and their use to infer the origin, relationships, or transmission route of a particular isolate 1 . One of the most important microbes are Fungi that are widely spread in the environment and cause many diseases for both human and animals. Some of them are zoonotic. Systemic infections occur when fungi get into the bloodstream and generally cause diseases that are more serious. Systemic fungal infections usually attack a person with a suppressed immune system, or by invasive fungus that is common in a certain geographic area. Genomics is increasing the power of microbial forensics through the design of gene-based diagnostic tests and directing analysis. The genomes of some of the important zoonotic pathogens (e.g. Salmonella typhimurium and Escherichia coli O157:H7) have been sequenced as what have been done with many important strains that might be used as biological agents threat (e.g. B. anthracis) 2,3 . Sequencing give an indication of the geographical area and source of infection in order to determine either the pathogen in a disease outbreak or poisoning bio-crime where a poisonous fungus is involved 4 . International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563 Vol.9, No.12, pp 417-421, 2016