Investigations and comparative detection ofCryptosporidiumspecies by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey

Abstract

A total of 70 water samples, including tap, river, fountain and well water were collected in the Ordu province, Middle Black Sea, Turkey and investigated for the detection of Cryptosporidium oocysts. The samples were directly screened microscopically for Cryptosporidium oocysts' detection by immunofluorescence test and subsequently DNA was extracted for the molecular detection by loop-mediated isothermal amplification (LAMP) and nested polymerase chain reaction (PCR). Eighteen out of the 70 (25.7%) water samples were found positive for Cryptosporidium spp. by immunofluorescence test and 19 (27.1%) were found positive by LAMP. Nested PCR products were not generated in any of the investigated water samples. A total of 16 randomly selected pellets were spiked with 10 Cryptosporidium oocysts to test the efficiency of the applied method. All the samples were found positive by LAMP for the presence of Cryptosporidium DNA, while the nested PCR assay was positive in only seven (43.75%) out of the 16 examined spiked samples. This is the first report on the occurrence of Cryptosporidium species in environmental and drinking water supplies in the Black Sea area.

Sensitive and Cost-Effective Detection ofToxoplasma Gondiiin Water Supplies of the Black Sea in Turkey by Loop-Mediated Isothermal Amplification (LAMP)

“…The high sensitivity of LAMP is because it uses six primers which target eight distinct internal regions on the target DNA, and can demonstrate as few as six copies of DNA (13). LAMP detection is more sensitive than PcR, in consensus with prior reports in fecal samples (1), in veterinary samples (17), and in environmental water samples (10,14).…”

“…Ten liters of water from each of the investigated sites were collected in sterile lastic bottles and were then transferred to the lab for analysis. All collected water samples were subjected to Al 2 (So 4 ) 3 flocculation and concentrated as previously described (9,10).…”

“…Briefly, PCR amplification was performed in 25 μL reaction mixtures containing 2. Nested PCR nested PcR targeting the 18S-rRnA gene was performed according to Kourenti and Karanis (11) and as later applied (10,12). In this study, the protocols were slightly modified.…”

LAMP DETECTION of Cryptosporidium SPECIES in SURFACE WATER SAMPLES COLLECTED from RIVER YESILIRMAK and STREAM TERSAKAN (Samsun-Amasya)

“…Five litters of water samples from investigated area were collected and they were flocculated with treatment in Aluminum Sulfate [Al2(SO4)3] and concentrated by sucrose gradient centrifugation for water pellets as described by Karanis and Kimura [6] and Kourenti et al [7] and Karanis et al [5] Koloren et al [8] …”

“…The S-adenosyl-L-methionine synthetase (SAM) LAMP method was performed on DNAs from water samples as previously described by Karanis et al [10], Bakheit et al [11], and Koloren et al [8]. C. parvum, C. hominis and C. meleagridis is amplified with the SAM-LAMP method as described before Bakheit et al [11], When we use this assay we can determine that one of these species is found in the sampling samples.…”