|
International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563 Vol.9, No.12, pp 417-421, 2016
|
Forensic molecular study using ITS region as a clue and possible fungal outbreak pre-warning indicator
Ali H. Al-Assadi1, Mohammad J. Al-Jassani2*
2University 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 isolate1. 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.
Fungal infection investigation is necessary in forensic cases that involve drugs and toxins analysis for both pre and postmortem cases since fungal metabolites can alter the drug chemical characteristics and make it undetectable or at least with false dose analysis results 5.
Very few studies manipulated fungal infection and toxins as a non-human DNA in forensic studies. Therefore, the aim of the present study was to evaluate a routine, high throughput, fast and sensitive molecular analysis in investigating fungal systemic infection using the PCR primers ITS1 and ITS4 that aid in human blood differentiation in some forensic cases. Add to that, to evaluate fungal infection load in animals that might act as source of an outbreak where a swift action is urgent to limit the scale of any outbreak and reassure the public when events are under control.
Sample collection
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). Pathogenic Aspergillus niger donated from the DNA research center, University of Babylon, was used as positive control.
Blood culture
Blood culture was done in triplicates and as following. About 0.5ml of blood were inoculated in 10ml Brain heart infusion broth (HIMEDIA) tubes then incubated at 37ºC for 24 hours. Thereafter, 0.5ml of culture broth was spread on Blood agar (HIMEDIA) plates and incubated at 37ºC for a week.
DNA extraction from blood
Genomic DNA was extracted from the whole blood (for individual and mixed blood samples) using Reagent genomic DNA Kit (Geneaid, Twain) according to the manufacturer instruction with modification by further protein cleanup using phenol/Chloroform (1:1).
DNA Extraction from fungi
Genomic DNA was extracted from Aspergillus niger isolated from phlegm of a patient, using Fungal/bacterial DNA Extraction Kit (ZYMO research, USA) and according to the manufacturer instructions.
Polymerase Chain Reaction (PCR)
PCR reactions were conducted using Thermal Cycler (Cleaver scientific, UK). The PCR (recipe and cycle) conditions were optimized in order to get the best possible yield utilizing about 40ng of the extracted DNA.
PCR Amplification of the ITS region
About 500-800bp of the internal transcribed spacer (ITS) region 6, of fungal rRNA genes was amplified using the fungal universal primers ITS1 as a forward primer and ITS4 as a reverse primer (Table 1) and as described by . The optimized recipe in Table (2) was followed and the PCR reaction was conducted using thermocycler (cleaver scientific, UK). The cycle conditions were as following, pre-denaturation at 95ºC for 1 min.; 35 cycles of (denaturation at 94ºC for 1 min, annealing at 50ºC for 1 min and extension at 72ºC for 1 min); final extension at 72ºC for 10 min followed by holding at 4ºC. A 5µl of the amplicon was electrophoresed through 1% agarose gel then examined and photographed using Bio-documentation/ Quantum ST5 (VILBER, France).
Table (1): Primers used in this study 7.
Name |
Sequence |
Tm(ºC) |
ITS1 |
5` TCCGTAGGTGAACCTGCGG 3` |
61 |
ITS4 |
5` TCCTCCGCTTATTGATATGC 3` |
55.3 |
Table (2): The optimized recipe components of a single 20µl PCR reaction.
Reagent |
Volume (µl) |
Final concentration |
Master mix (i-Taq, Intron) |
|
|
Water (sterile, nuclease free) |
11 |
|
Primer ITS4 (10 mM) |
2 |
1 mM |
Primer ITS1 (10 mM) |
2 |
1 mM |
DNA template (20-40 ng/ml) |
5 |
1-2 ng/ml |
Total |
20 |
|
Results and Discussion:
Blood Culture
Blood culture has showed no results even when Blood agar plates were incubated for a week. This is expected since many fungal strains need weeks to appear in culture. Add to that, positive blood culture results were obtained in less than 50% of clinical cases 8,9.
PCR
PCR result shows that mycosis in human leukemia patient 70% in comparison with 30% in non-symptomatic samples which give an indication for high susceptibility of leukemic people for being infected due to immune suppression. Detection of fungus in 30% of apparently healthy people give an indication of a possible disease outbreak either from contact animals like chicken and sheep were 40% and 20% found infected respectively or other sources, especially with a high percentage of human positive results in comparison with animal results (Table 3) and (Figure 1).
With the PCR amplicon gel electrophoresis results (Figure 2), we have found that a (1:1) mixture of positive (H1) and negative (H2) human blood sample show a positive band (lane 4). This result indicates the presence of H1 blood in the mix and can be used as a supportive clue with involvement of H1 in the forensic case.
Table (3): PCR results of the tasted blood sample
Positive results (%) |
Negative |
Positive |
Sample |
30 |
14 |
6 |
Human |
70 |
3 |
7 |
Human Leukaemia Patient |
40 |
12 |
8 |
Chicken |
20 |
16 |
4 |
Sheep |
Figure (1): PCR results of the tested blood samples showing the mycosis percentage.
~540bp
Figure (2): Agarose gel electrophoresis of PCR amplicon. L: DNA size marker. 1: YF (Positive Control), 2: HF3, 3: SH2, 4: H1+H2, 5: CH2, 6: CH3, 7: H2, 8: H3, 9: H1, 10 (negative control) Electrophoresis conditions: Agarose concentration 1 %, power applied: 100 V (7mA / cm), time of run: 45 min. staining method; precast red fast.
Fungal infection investigation is necessary in forensic cases that involve drugs and toxins analysis for both pre and postmortem cases since fungal metabolites can alter the drug chemical characteristics and make it undetectable or at least with false dose analysis 5,10.
The authors wish to express their thanks to Miss. Takwa S. Al-Meamar for her assistance.
References
1. Cummings, C. A. and Relman, D. A. Genomics, and microbiology Microbial forensics cross-examining pathogens. Science, 2002, 296: 1976-9.
2. American Heritage. Dictionary of the English language, 4th Ed. The American Heritage®, Boston. 2003, Available at: http://www.thefreedictionary.com/forensics
3. Budowle, B., Schutzer, S. E., Ascher, M. S., Atlas, R. M., et. al. Toward a system of microbial forensics: from sample collection to interpretation of evidence. Appl. environ. Microbiol., 2005, 71 (5): 2209-2213.
.4 Kingsley, M. T., Straub, T. M., Call, D. R., Daly, D. S., Wunschel, S. C., Chandler, D. P. Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays. Appl Environ Microbiol., 2002, 68: 6361-70.
5. Martínez-Ramírez, J. A., Strien, J., Walther, G. and Peters, F. T. Search for fungi-specific metabolites of four model drugs in postmortem blood as potential indicators of postmortem fungal metabolism. Forensic Sci. Int., 2016, 262:173-8.
6 Embong, Z., Wan Hitam Wan H., Yean, C. Y., Abdul Rashid N. H., et. al. Specific detection of fungal pathogens by 18S rRNA gene PCR in microbial keratitis. BMC Ophthalmol., 2008, 8:7.
7. White, T. J., Bruns, T., Lee, S. and Taylor, J. W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications, eds. Innis, M. A.; Gelfand, D. H.; Sninsky, J. J. and White, T. J. Academic Press Inc., New York. pp., 1990, 315-322.
8. Jamal, W., Tamaray, G., Pazhoor, A., Rotimi, V. O. Comparative evaluation of BacT/ALERT 3D and BACTEC systems for the recovery of pathogens causing bloodstream infections. Med Princ Pract., 2006, 15:223-227.
9. Zieliński, A. and Czarkowski, M. P. Infectious diseases in Poland in 2007. Przegl Epidemiol., 2009, 63:161-167.
.01 Martinez-Ramirez, J. A. Studies on drug metabolism by fungi colonizing decomposing human cadavers. Part I: DNA sequence-based identification of fungi isolated from postmortem material. Anal Bioanal Chem., 2013, 405: 8443-50.
*****
Extra Page not to be Printed out.
For your Research work, for citations/References Log on to=
www.sphinxsai.com
International Journal of ChemTech Research
International Journal of PharmTech Research 101513130
Sai Scientific Communications
*****
Extra Page not to be Printed out.
For your Research work, for citations/References Log on to=
www.sphinxsai.com
International Journal of ChemTech Research
International Journal of PharmTech Research 101513130
Sai Scientific Communications
*****