Patent ID: 7427483

Claim:
A procedure using specific biotin labeled nucleotide probes directed to the highly conserved 42-kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP1) gene as a target for the quantitative determination of Plasmodium falciparum DNA, comprising: (a) a procedure for amplifying the 42-kDa C-terminal region of the MSP1 gene from negative control (non-infected) and P. falciparum infected samples by polymerase chain reaction (PCR) comprising the following steps: (i) isolating P. falciparum DNA from the negative control and P. falciparum infected samples; and (ii) PCR amplifying the DNA of step (a)(i) in the presence of digoxigenin-11-dUTP using the oligonucleotides SEQ ID NO: 1 and SEQ ID NO: 2 under the following PCR conditions: denaturing at 94° C. for 1 minute; annealing at 55° C. for 2 minutes; elongating at 72° C. for 1 minute each cycle, for 20 cycles; thereby producing PCR products in which the 42-kDa C-terminal region of the MSP1 gene amplified from said samples is conjugated to digoxigenin-11-dUTP; (b) a procedure for the synthesis of three specific biotin labeled nucleotide probes 7, 8, and 9 directed respectively to three different fragments of 420 bp (fragment 1), 420 bp (fragment 2), and 379 bp (fragment 3) located in the 42-kDa C-terminal region of the MSP1 gene of P. falciparum comprising the following steps: (i) performing a PCR reaction using the 42-kDa C-terminal region of the P. falciparum MSP1 gene as template and the oligonucleotides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 as primers, under the following PCR conditions: denaturing at 94° C. for 1 minute; annealing at 65° C. for 2 minutes; elongating at 72° C. for 1 minute each cycle, for 35 cycles; wherein SEQ ID NO: 1 and SEQ ID NO: 3 amplify fragment 1, SEQ ID NO: 4 and SEQ ID NO: 5 amplify fragment 2, and SEQ ID NO: 2 and SEQ ID NO: 6 amplify fragment 3; (ii) ligating amplified fragments 1, 2, and 3 produced in step (b)(i) into the pCR R 2.1 plasmid vector and introducing ligation products into E. coil strain TOP10F′, thereby producing vectors 4, 5, and 6 comprising fragments 1, 2, and 3, respectively; (iii) isolating SpeI-XhoI fragments containing fragments 1, 2, and 3 from vectors 4, 5, and 6 respectively; and (iv) synthesizing three specific biotin labeled nucleotide probes 7, 8, and 9 using the SpeI-XhoI fragment isolated from vector 4 as template and SEQ ID NO: 1 and SEQ ID NO: 3 as primers to synthesize probe 7, the SpeI-XhoI fragment isolated from vector 5 as template and SEQ ID NO: 4 and SEQ ID NO: 5 as primers to synthesize probe 8, and the SpeI-XhoI fragment isolated from vector 6 as template and SEQ ID NO: 2 and SEQ ID NO: 6 as primers to synthesize probe 9; and (c) using the PCR products produced in (a) and probes 7, 8, and 9 synthesized in (b) in an enzyme linked immunosorbent assay (ELISA) to quantitatively determine the 42-kDa C-terminal region of the MSP1 gene in said samples, and thereby quantitatively determining P. falciparum DNA in said samples.