Multiplex quantitative PCR
a multi-quantity, multi-quantity technology, applied in the direction of drug compositions, cardiovascular disorders, extracellular fluid disorders, etc., to achieve the effect of diagnosing the subject at higher cardiovascular risk
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example 1
[0237]Each PCR reaction was carried out in a total volume of 10 μL per well of a standard 384 well assay plate. The standard reaction mix contained the following components: 5 ng target DNA, 1.0 μM EvaGreen® Dye (Biotium, Hayward, Calif.), 300 nM Tel G modified primer, 300 nM Tel C modified primer, 300 nM B2M-F primer, 300 nM 82M-R primer, 100 nM B2M probe, 1× RNase P Mix (TaqMan® Copy Number Reference Assay RNase P, Thermo Fisher Scientific, Inc.), 1× Quantifast Probe PCR Master Mix (QIAGEN, Inc., Germantown, Md.). Table 2 below provides various primer sequences.
TABLE 2LengthSEQOligo(nucleotides)ID NO.SequenceTel G4515′-ACACCTCCTCCATGGTTTGGGTTTGmodifiedGGTTTGGGTTTGGGTTAGTG-3′Tel C4325′-TGTTAGCGACGCGATATCCCTATCC-3′modifiedCTATCCCTATCCCTAACA-3′B2M-F2235′-CCAGCAGAGAATGGAAAGTCAA-3′B2M-R2845′-TCTCTCTCCATTCTTCAGTAAGTCAACT-3′B2M-P*2755′-ATGTGTCTGGGTTTCATCCATCCGACA3-3′
[0238]The standard cycling conditions for the disclosed triplex qPCR assay are those shown in Table 3.
TABLE 3Cycli...
example 2
t of the Effect of Tel G Modified and Tel C Modified Primer Concentration
[0240]The standard reaction conditions described above were used, except that the concentration of the Tel G modified and Tel C modified were varied. The following concentrations were examined: 400 nM Tel G modified and 400 nM Tel C modified; 300 nM Tel G modified and 100 nM Tel C modified; 600 nM Tel G modified and 100 nM Tel C modified; 300 nM Tel G modified and 300 nM Tel C modified; and 600 nM Tel G modified and 300 nM Tel C modified. The melting curves for the reactions with the foregoing Tel G modified / Tel C modified primer concentrations are shown in FIG. 2A-FIG. 2F. The data show that when the reaction was carried out with 300 nM Tel G modified and 300 nM Tel C modified, all three targets have similar amplification amplitude, suggesting that all three PCR reactions generate approximately similar amounts of products and the assay reaches the desired balance for the three targets. Comparable amounts of th...
example 3
tion Efficiency
[0241]An 8-point 2-fold serial dilution of the Mosaic Male genomic DNA was used to calculate the PCR efficiencies. The DNA concentration in the final PCR reaction for each point is shown in Table 5. The PCR efficiencies of each of the target for each primer combination tested were obtained with absolute quantification method in the Roche LC480 program and are summarized in Table 6.
TABLE 5Final concentrationStandard pointin PCR (ng / μl)Std15Std22.5Std31.25Std40.625Std50.3125Std60.1563Std70.0781Std80.0391
TABLE 6PCR Amplification EfficienciesTRNasePB2MT only104.0%——S only—98.6%96.3%300 nM TelG97.5%106.8%97.4%100 nM TelC300 nM TelG95.4%107.9%96.3%300 nM TelC400 nM TelG97.4%105.9%96.6%400 nM TelC600 nM TelG95.1%105.2%97.5%100 nM TelC
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