Loopamp la 320c

Wizard Genomic DNA Purification kits were purchased and used to extract DNA (A1125; Promega, USA). DNA Isothermal Amplification kits (HT0600; HuiDeXin, China) and AapCas12b nuclease (HT100008; HuiDeXin, China) were purchased and used for LAMP and CRISPR-top assay. A real-time turbidimeter (Loopamp LA-320c; Eiken, Japan) was used to monitor the LAMP products. A real-time fluorescence qPCR instrument (QuantStudio 6 Flex; Applied Biosystems, USA) was used as the fluorescence reader. MALDI-TOF MS (microflex LRF; Bruker, Germany) was used to identify the isolated strains.

Wizard Genomic DNA purification kits (catalog number A1125; Promega, Madison, WI, USA) were used for DNA extraction from pure cultures and clinical specimens. DNA isothermal amplification kits (catalog number HT0600; HuiDeXin, Tianjin, China) were used for the LAMP reaction and the P. aeruginosa CRISPR-top assay. AapCas12b enzyme with Cas12b buffer (catalog number HT100008; HuiDeXin) was used for gRNA/Cas12b complex preparation. Premix Ex Taq 2× (probe qPCR, catalog number RR390A; TaKaRa, Dalian, China) was used for qPCR detection. A spectrophotometer with fixed wavelengths of 260 and 280 nm (Nanodrop LITE; Thermo Fisher Scientific, Shanghai, China) was used to determine the concentration of double-stranded DNA. A real-time turbidimeter (Loopamp LA-320c; Eiken, Tokyo, Japan) was used to monitor the LAMP products in primer screening. An isothermal metal bath (catalog number 88870004; Thermo Fisher Scientific) was used for gRNA/Cas12b complex preparation. A real-time fluorescence qPCR instrument (QuantStudio 6 Flex; Applied Biosystems, Foster City, CA, USA) was used for qPCR and as the fluorescence reader in the CRISPR-top reaction. MALDI-TOF MS (microflex LRF; Bruker, Bremen, Germany) was used to identify the strains isolated from clinical samples.

A 25 μL LAMP reaction volume contained 12.5 μL 2× isothermal reaction buffer, 1 μL Bst 2.0 DNA polymerase (8U), 0.4 μL LAMP primer premixture, 1 μL DNA template, and DNase/RNase-free deionized water (DW) up to 25 μL. The LAMP reaction was conducted at 56°C for 1 h using a real-time turbidimeter containing a thermo block (Loopamp LA-320c; Eiken, Japan), and the turbidity of the reaction mixture was captured by the turbidimeter in real time.