Isolation and identification of algae
Five cyanobacterial isolates were derived from the Tigris River, including Microcystis flosaquae, Microcystis sp, anabaena circinalis, nostoc commune, and westiellopsis prolifica, which contributed to four cyanobacterial orders: Chroococales, Stigonematales, and Nostocales, as well as one chlorophyceae isolate, Chlorella vulgaris where used as negative control test.
Extraction of DNA algae
Genomic DNA was successfully extracted from samples( algae ) by Easy Pure® Genomic DNA Kit , Gel electrophoresis was used to validate and analyze the DNA bands. as in (Fig. 1)
Detection of cyanobacteria by PCR test
A phycocyanin operon gene fragment from cyanobacteria containing the IGS (cpcBA-IGC) was amplified. When all of the DNA extracts with a size of 650 bp were analyzed in gel electrophoresis (Fig. 2), separate amplicon patterns were generated, confirming the presence of cyanobacterial DNA from samples obtained from fresh water of the Tigris River in Baghdad. Whereas the lane of a green alga, Chlorella vulgaris, lacks the Pycocyanin operon, no PCR product was obtained, indicating the high specificity of the primers used. The findings confirmed the presence of cyanobacterial DNA in isolates taken from the Tigris river in Baghdad city and successfully amplified a fragment usingPCβ-PCα primes set for cyanobacterial detection, and the same results were reported in study of (13).
Five different cyanobacterial species were identified, and these potentially cyanotoxin-producing species were found in Tigris river samples, such as Microcystis flosaquae, Microcystis sp, Anabaena circinalis, Nostoc commune and Westiellopsis prolifica. The common cytotoxin that blocks protein synthesis, with renal and liver failure as the first clinical symptoms, has a biologically active alkaloid found in several cyanotoxin producing species.
Detection of Cylidrospemopsin by PCR assay
in this study, Traditional PCR has been used to identify cyanobacteria that produce cylidrospemopsin and possess an aminotransferase enzyme. The (CKc-F/R) primers successfully amplified the 325bp fragments of the aoaC gene from all microcystin-producing cyanobacterial isolates. With the exception of Chlorella vulgaris, (Fig. 3). The specificity of 325bp primers showed to be highly specific for samples forming cylidrospemopsin ,since there was no DNA amplified from chlorophyta used in this study., The findings corroborated the findings of (14).
PCR methods were used in several experiments to find the cylindrospermopsin producers in freshwater. While bio-molecular detection methods have gained popularity only a few studies have focused on using PCR methods to rapidly track cylindrospermopsin producers in river water due to their specificity and speed. Marbun et al., (15) have shown that the qPCR approach can be used to detect C. raciborskii in reservoirs quickly and on-site. Furthermore, the findings indicate that cylindrospermopsin is a major cyanotoxin in Kinmen Island's reservoirs.
Detection of Saxitoxin by PCR assay
The sxtA-F/R set primers were used to detect the sxtA gene, which is unique to cyanobacteria that produce saxitoxin and amplified 125bp. the results showed that the sxtA gene was found in all samples isolated from the Tigris River Since no DNA from chlorophyta was amplified (figure 4). The findings were in line with those of many other studies. (16,17).
Using traditional PCR, the sxtA-F/R set primer designed by (18) was seen to be specific to saxitoxin-producing Anabaena circinalis. The sxtAF/R primers also amplified the sxtA gene from other cyanobacteria that produce saxitoxin. Saxitoxins (STXs) cause neurotoxicity by blocking sodium ion channels in nerve axon membranes; STXs are also known to block calcium channels. STXs also affect heart muscle cells by prolonging potassium channel gating, which can result in changes in ion influx into the cell. (19,20). To generate fresh portable water, most existing drinking water treatment plantes (WTPs) use traditional treatment methods such as coagulation-flocculation, sedimentation, sand filtration, and disinfection (21).
The increased incidence of cyanobacterial blooms in freshwater bodies in several decades has prompted water management agencies to pay closer attention to the dangers posed by toxic cyanobacteria. While reports of neurotoxins are becoming more common, toxic events are still mainly diagnosed using microcystins. (22).