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A Simple and Rapid DNA Purification Method for Detection of Leishma- nia DNA in Peripheral Blood of Patients with Visceral Leishmaniasis


1 Instructor, Department of Microbiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
2 Associate Professor, Department of Molecular and clinical Virology, School of paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
*Corresponding Author: A Behzad-Behbahani, School of Paramedical Sciences, Meshkinfam Street, Shiraz, Iran, Tel.: +98-7112295023, Fax: +98-7112289113, E-mail: [email protected].
Shiraz E-Medical Journal. 9(2): 75-81.
Article Type: Research Article; Received: Jan 12, 2008; epub: Apr 1, 2008;

Abstract


Introduction: Recently polymerase chain reaction technique is being used for detection of leishmania DNA in clinical samples of patients suspected with visceral leishmaniasis. Since many of the patients are children and also suffering from anemia and leucopenia, there is a need for the development of a DNA extraction method using small amount of clinical samples. In the present study, a simple and rapid method was introduced for performing the DNA extraction procedure which was capable of removing inhibitors from the whole blood sam- ples and of providing the appropriate DNA for Leishmania PCR amplification.

Materials and Methods: EDTA-peripheral blood specimens were collected from healthy volunteers and frozen at -20°C. The thawed specimens were spiked with a definite number of L. infantum promastigotes. Using in house made lysis buffer and boiling, a DNA extraction method was developed for the purification of Leishmania genome from 500 IJl of blood samples. The developed method was then compared with the classical standard method to obtain the highest sensitivity for the diagnosis of visceral leishmaniasis.

Results: The sensitivity of the PCR for the developed method was 10 copies of L. infantum DNA per reaction tube. When clinical samples collected from patients with a definite diagnosis of visceral leishmaniasis were used, no false negative was demonstrated with newly developed method.

Conclusion: Since hazardous materials such as phenol-chloroform are not used during extraction procedure, it seems to be much safer than the standard phenol-chloroform method. However, the use of small amount of sample (i.e. 0.5 IJl) and removal of PCR-inhibitors from the whole blood are the major advantages of our devel- oped DNA extraction method.

Keywords: Leishmania, DNA purification, Peripheral blood.