Iplex enzyme

MassArray was used to identify MTB and detect MTB resistance gene mutation sites. The anti-tuberculosis drugs and their corresponding gene resistance loci are shown in Table 1. Two PCR reaction systems were performed simultaneously. The reaction system contains HPLC H₂O 0.8 μL, 10XPCR buffer with 20 mM MgCl₂ 0.5 μL, 25 mM MgCl₂ 0.4 μL, 25 mM dNTP Mix 0.1 μL, 0.5 μM Primer Mix 1 μL, PCR Enzyme 0.2 μL and 2 μL template DNA, in a final volume of 5 μ. The reaction program was: 95°C for 2 min; 95°C for 30s, 60°C for 30s, 72°C for 60s, for 45 cycles; 72°C for 5 min, hold at 4°C. Add 2 μL shrimp alkaline phosphatase (SAP) in each well, 37°C for 40 min, 85°C for 5 min, hold at 4°C. Add 2 μL iPLEX extension mix (nanopure water 0.62 μL, iPLEX buffer 0.2 μL, iPLEX termination mix 0.2 μL, extend primer mix 0.94 μL, and iPLEX enzyme 0.04 μL, Agena Bioscience, San Diego, CA) in each well. The reaction program was: 95°C for 30s; 95°C for 5 s, (52°C for 5 s, 80°C for 5 s, for 5 cycles), for 40 cycles; 72°C for 3 min, hold at 4°C.
The production was then carried out at Zhejiang Digena R&D Center, on a high-throughput MassARRAY platform with data analyzed using Typer 4.0 and plate manager 1.0 software. The quality of the test results was classified as No-Alleles, Low Probability, Aggressive, Moderate, Conservative.