Detecting its1 in toxoplasma gondii and neospora caninum using pcr

摘要

Method of detecting a protozoan parasite in a sample containing the parasite, the method comprising the steps of: (a) adding to the sample a pair of flanking oligonucleotide primers, at least one flanking primer being specific for and each being complementary to an opposite strand of a double stranded DNA molecule encoding the ITS1 of the protozoan parasite and flanking a region of the ITS1; (b) further adding to the sample a pair of nested oligonucleotide primers, each nested primer being specific for and complementary to an opposite strand of the DNA encoding the ITS1 of the protozoan parasite, the nested primers being complementary to the region of the ITS1 spanned by the flanking primers; (c) providing buffers, reagents, nucleotides and a thermostable DNA polymerase to the sample to form a reaction mixture; (d) heating the sample to a temperature such that the double stranded DNA encoding the ITS1 of the protozoan parasite denatures to form single stranded DNA molecules; (e) cooling the denatured sample to a temperature such that only the flanking primers anneal to their respective complementary sequences on the denatured DNA molecules; (f) heating the denatured and annealed sample to a temperature such that the DNA polymerase extends the primers to form new double stranded DNA molecules spanning the region of the ITS1 defined by the flanking primers; (g) repeating steps (d), (a) and (f) such that the number of copies of the region of DNA encoding the ITS1 region is amplified; (h) heating the sample to a temperature such that the newly amplified double stranded DNA encoding the ITS1 region denatures to form single stranded DNA molecules; (i) cooling the denatured sample to a temperature such that only the nested primers anneal to their respective complementary sequences on the denatured DNA; (j) heating the denatured and annealed sample to a temperature such that the DNA polymerase extends the primers to form new double stranded DNA molecules spanning the region of the ITS1 defined by the nested primers; (k) repeating steps (h), (i) and (j) such that the number of copies of the region of DNA is amplified; and (l) detecting the amplified DNA.