DNA extraction from hydatid cyst protoscolices: Comparison of five different methods
Aim: The current study aimed to find out a simple, practical and high throughput DNA isolation method for extraction of DNA from hydatid cyst samples. Materials and Methods: Cattle and sheep isolate of hydatid cysts were obtained from the slaughterhouse, and hydatid fluid and protoscolices were c...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Veterinary World
2018-02-01
|
| Series: | Veterinary World |
| Subjects: | |
| Online Access: | http://www.veterinaryworld.org/Vol.11/February-2018/23.pdf |
| _version_ | 1818923672735318016 |
|---|---|
| author | Afshin Barazesh Bahador Sarkari Sepideh Ebrahimi Mehdi Hami |
| author_facet | Afshin Barazesh Bahador Sarkari Sepideh Ebrahimi Mehdi Hami |
| author_sort | Afshin Barazesh |
| collection | DOAJ |
| description | Aim: The current study aimed to find out a simple, practical and high throughput DNA isolation method for extraction of DNA from hydatid cyst samples.
Materials and Methods: Cattle and sheep isolate of hydatid cysts were obtained from the slaughterhouse, and hydatid fluid and protoscolices were collected in a sterile condition. Protoscolices were washed, 3 times with phosphate buffered saline, and DNA was extracted by different methods including manual extraction with freeze/thawing and phenol-chloroform, Triton X-100 extraction, and by a commercial kit (YTA, Yekta Tajhiz Azma, Iran) with three different modifications in the kit's manufacturer instructions. The obtained DNA from the different methods was evaluated by Nanodrop in terms of the yield of DNA and carbohydrates or protein contaminations. To compare the quality of the extracted DNA, two pieces of the mitochondrial genome of Echinococcus granulosus, cox1, and nad1, were polymerase chain reaction (PCR)-amplified, using each of the DNA prepared by different methods. Electrophoresis of PCR products was carried out on the agarose gel.
Results: The DNA extracted by manual method, using phenol/chloroform, had the highest yield, yet with the highest level of protein and carbohydrate contamination. The DNA extracted using two-step incubations, initially at 60°C for 2 h and then overnight at 37°C, was the most purified DNA with the lowest rate of contamination.
Conclusion: Findings of the study demonstrated that modification in the currently available commercially DNA extraction kit resulted in the development of a high throughput DNA isolation method. This method can be recommended for the extraction of DNA from hydatid cysts, especially the cattle isolate where the extraction of DNA in these samples are usually problematic. |
| first_indexed | 2024-12-20T02:13:10Z |
| format | Article |
| id | doaj.art-aeea6db04c134bc5a99a43789259a6d7 |
| institution | Directory Open Access Journal |
| issn | 0972-8988 2231-0916 |
| language | English |
| last_indexed | 2024-12-20T02:13:10Z |
| publishDate | 2018-02-01 |
| publisher | Veterinary World |
| record_format | Article |
| series | Veterinary World |
| spelling | doaj.art-aeea6db04c134bc5a99a43789259a6d72022-12-21T19:57:00ZengVeterinary WorldVeterinary World0972-89882231-09162018-02-0111223123410.14202/vetworld.2018.231-234DNA extraction from hydatid cyst protoscolices: Comparison of five different methodsAfshin Barazesh0Bahador Sarkari1Sepideh Ebrahimi2Mehdi Hami3Student Research Committee, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; The Persian Gulf Tropical Medicine Research Centre, Bushehr University of Medical Sciences, Bushehr, Iran.Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.Technical Deputy of East-Azarbaijan Province, Veterinary Directorate, Iran Veterinary Organization, Iran.Aim: The current study aimed to find out a simple, practical and high throughput DNA isolation method for extraction of DNA from hydatid cyst samples. Materials and Methods: Cattle and sheep isolate of hydatid cysts were obtained from the slaughterhouse, and hydatid fluid and protoscolices were collected in a sterile condition. Protoscolices were washed, 3 times with phosphate buffered saline, and DNA was extracted by different methods including manual extraction with freeze/thawing and phenol-chloroform, Triton X-100 extraction, and by a commercial kit (YTA, Yekta Tajhiz Azma, Iran) with three different modifications in the kit's manufacturer instructions. The obtained DNA from the different methods was evaluated by Nanodrop in terms of the yield of DNA and carbohydrates or protein contaminations. To compare the quality of the extracted DNA, two pieces of the mitochondrial genome of Echinococcus granulosus, cox1, and nad1, were polymerase chain reaction (PCR)-amplified, using each of the DNA prepared by different methods. Electrophoresis of PCR products was carried out on the agarose gel. Results: The DNA extracted by manual method, using phenol/chloroform, had the highest yield, yet with the highest level of protein and carbohydrate contamination. The DNA extracted using two-step incubations, initially at 60°C for 2 h and then overnight at 37°C, was the most purified DNA with the lowest rate of contamination. Conclusion: Findings of the study demonstrated that modification in the currently available commercially DNA extraction kit resulted in the development of a high throughput DNA isolation method. This method can be recommended for the extraction of DNA from hydatid cysts, especially the cattle isolate where the extraction of DNA in these samples are usually problematic.http://www.veterinaryworld.org/Vol.11/February-2018/23.pdfDNA extractionhydatid cystprotoscolices |
| spellingShingle | Afshin Barazesh Bahador Sarkari Sepideh Ebrahimi Mehdi Hami DNA extraction from hydatid cyst protoscolices: Comparison of five different methods Veterinary World DNA extraction hydatid cyst protoscolices |
| title | DNA extraction from hydatid cyst protoscolices: Comparison of five different methods |
| title_full | DNA extraction from hydatid cyst protoscolices: Comparison of five different methods |
| title_fullStr | DNA extraction from hydatid cyst protoscolices: Comparison of five different methods |
| title_full_unstemmed | DNA extraction from hydatid cyst protoscolices: Comparison of five different methods |
| title_short | DNA extraction from hydatid cyst protoscolices: Comparison of five different methods |
| title_sort | dna extraction from hydatid cyst protoscolices comparison of five different methods |
| topic | DNA extraction hydatid cyst protoscolices |
| url | http://www.veterinaryworld.org/Vol.11/February-2018/23.pdf |
| work_keys_str_mv | AT afshinbarazesh dnaextractionfromhydatidcystprotoscolicescomparisonoffivedifferentmethods AT bahadorsarkari dnaextractionfromhydatidcystprotoscolicescomparisonoffivedifferentmethods AT sepidehebrahimi dnaextractionfromhydatidcystprotoscolicescomparisonoffivedifferentmethods AT mehdihami dnaextractionfromhydatidcystprotoscolicescomparisonoffivedifferentmethods |