2.3. MADCAT Procedures
Up to 12 μL sample (plasmid, gDNA, whole blood, serum or tissue
homogenates) was lysed with 16.7
μL
of 3 × lysis mixture, 0.5 μL of capture probes (0.1 μM), 2.5 μL of
proteinase K (20 mg/mL, Tiangen), and 18.3 μL of deionized water at 56 ℃
for 5 min with vigorous shaking in customized 96-well capture plate.
Thermal denaturation was performed at 98 ℃ for 5 min, followed by 10-min
target capture at 55 ℃. Both the lysis and capture processes were
carried out under the condition of shaking at 1200 rpm with 96-well
Thermomixer (Eppendorf), while the denaturation was performed in a
96-well PCR thermocycler. After washing three times with washing
solution to remove all unbound probes and irrelevant nucleic acids, the
captured targets were then amplified with a 25 μL PCR reaction mix
containing 0.2 µM of each primer, 0.1 µM of Taqman probe (VP-FP), and
12.5 μL of 2 × probe qPCR premix (Takara). The PCR reaction was carried
out on the CFX96 Real-Time PCR Detection System (Bio-Rad) or
LightCycler® 96 System (Roche Life Science), with 95 ℃ for 30 s,
followed by 40 cycles of 95 ℃ 5 s, 54 ℃ 10 s and 72 ℃ 20 s. A positive
result was called if the Ct value < 40 and a sigmoidal plot is
observed.