Muhammad Asif Muieed
Comparative Studies On The Sensitivity Of Polymerase Chain Reaction (Pcr) And Microscopic Examination For The Detection of Trypanosoma Evansi in Horses - 2005
The polymerase chain reaction (PCR) was standardized and its efficacy was evaluated against microscopic examination i.e. Giemsa stained smear method ['or the diagnosis of Trypanosoma evansi infection (Surra) in horses. l3lood samples were collected from 100 suspected horses from different localities in Lahore.
Under aseptic precautions blood smears were prepared, after drying and fixing with methanol, slides were stained by Giemsa stain method of staining. By stained blood smear method 5 out of 100 horses were found positive For T. evansi infection. The polymerase chain reaction (PCR) was carried out on the blood of' the same suspected horses to evaluate its efficacy in the diagnosis of' T. evansi infection and to compare its diagnostic value against the microscopic examination method currently in use. For this purpose total genomic DNA was extracted from suspected blood samples. The PCR reaction was performed in a 50tl reaction mixture containing I X Taq BuFfer, 0.2 mM dNTP Mixture. I .5 mM MgCIl2 2.5 U/1i1 Taq Polymerase. 4uM of' each primer, 2 ul of DNA extracted and 31.5 p1 of DNase - free deionised water. The tubes containing the mixture were subjected to 30 cycles of amplification in a thermocycler. During each cycle the sample of' DNA was denatured at 93° C' For 30 seconds, annealed at 45° C For 30 seconds and extended at 720 C For I minute. Prior to the cycling and at the end of' cycling the mixture was subjected to incubation at 93° C for a period of 3 minutes and final extension at 72° C for a period of 5 minutes, respectively. PCR product was then characterized by 2.5% of agarose gel electrophoresis. To confirm the presence of DNA and to estimate its size it was compared with a DNA ladder and was photographed with a Polaroid camera. The polymerase chain reaction (PCR) revealed 16 positive cases out of 100 above mentioned suspected cases. These 16 positive cases diagnosed by polymerase chain reaction (PCR) also included animals, which were diagnosed by stained blood smear method.
It can be concluded that polymerase chain reaction (PCR) is a superior and sensitive (16%) in comparison with the microscopic examination i.e. Giemsa stained smear method (5%). Polymerase chain reaction (PCR) is more effective in cases where the parasitemia is low and this test could be used in other species of animals especially camels where the disease is more chronic and difficult to confirm by. other routine methods. PCR would not only ensure early diagnosis and treatment in individual animals but can detect animal reservoirs of infection and would help to eliminate threat to equine and camel herds which are grazed and housed together and where blood sucking mechanical fly vectors are ever present.
Department of Pathology
0860,T
Comparative Studies On The Sensitivity Of Polymerase Chain Reaction (Pcr) And Microscopic Examination For The Detection of Trypanosoma Evansi in Horses - 2005
The polymerase chain reaction (PCR) was standardized and its efficacy was evaluated against microscopic examination i.e. Giemsa stained smear method ['or the diagnosis of Trypanosoma evansi infection (Surra) in horses. l3lood samples were collected from 100 suspected horses from different localities in Lahore.
Under aseptic precautions blood smears were prepared, after drying and fixing with methanol, slides were stained by Giemsa stain method of staining. By stained blood smear method 5 out of 100 horses were found positive For T. evansi infection. The polymerase chain reaction (PCR) was carried out on the blood of' the same suspected horses to evaluate its efficacy in the diagnosis of' T. evansi infection and to compare its diagnostic value against the microscopic examination method currently in use. For this purpose total genomic DNA was extracted from suspected blood samples. The PCR reaction was performed in a 50tl reaction mixture containing I X Taq BuFfer, 0.2 mM dNTP Mixture. I .5 mM MgCIl2 2.5 U/1i1 Taq Polymerase. 4uM of' each primer, 2 ul of DNA extracted and 31.5 p1 of DNase - free deionised water. The tubes containing the mixture were subjected to 30 cycles of amplification in a thermocycler. During each cycle the sample of' DNA was denatured at 93° C' For 30 seconds, annealed at 45° C For 30 seconds and extended at 720 C For I minute. Prior to the cycling and at the end of' cycling the mixture was subjected to incubation at 93° C for a period of 3 minutes and final extension at 72° C for a period of 5 minutes, respectively. PCR product was then characterized by 2.5% of agarose gel electrophoresis. To confirm the presence of DNA and to estimate its size it was compared with a DNA ladder and was photographed with a Polaroid camera. The polymerase chain reaction (PCR) revealed 16 positive cases out of 100 above mentioned suspected cases. These 16 positive cases diagnosed by polymerase chain reaction (PCR) also included animals, which were diagnosed by stained blood smear method.
It can be concluded that polymerase chain reaction (PCR) is a superior and sensitive (16%) in comparison with the microscopic examination i.e. Giemsa stained smear method (5%). Polymerase chain reaction (PCR) is more effective in cases where the parasitemia is low and this test could be used in other species of animals especially camels where the disease is more chronic and difficult to confirm by. other routine methods. PCR would not only ensure early diagnosis and treatment in individual animals but can detect animal reservoirs of infection and would help to eliminate threat to equine and camel herds which are grazed and housed together and where blood sucking mechanical fly vectors are ever present.
Department of Pathology
0860,T