Reagents and equipment. The magnetic bead virus nucleic acid extraction kit was purchased from Suzhou Tianlong Bio-Technology Co. Ltd. The reverse transcriptase was from Vazyme Biotech Co. Ltd. (Nanjing), and the Taq DNA polymerase was from Fapon Biotech Inc. (Zhuhai). The Power SYBR Green PCR Master Mix Kit was from ABI Co.Ltd. (USA) .OneStep ABI instrument (USA).The primers and probes used in common epidemic swine diseases detection were synthesized by Genscript (Nanjing) Co., Ltd, and the detailed sequences are shown in Appendix Table 1. The microfluidic PCR analyzer was purchased from Shenzhen Shineway Technology Corporation and the fluorescence quantitative PCR instrument was from Hangzhou Bioer Technology Co. Ltd.
Nucleic acid extraction and amplification. The nucleic acid of the pathogenic microorganisms were extracted following the instructions of the magnetic bead nucleic acid extraction kit. For synthesis of cDNA, 16μL master mix consisted of RT-PCR buffer, dNTPs, random hexamer, reverse transcriptase enzyme and RNase inhibitor was mixed with 24μL of extracted RNA. Reverse transcripyion was performed at 37℃ for 1 hour. The extracted DNA solution was used as the template solution for the subsequent experiments, and was stored at -20 ° C in a refrigerator; or at 4°C in a refrigerator or on ice in case of immediate use in a subsequent experiment. In the nucleic acid amplification, a 25 μLreaction solution was prepared with the following components: 5 μLtemplate solution, 0.5 μLforward primer (10 μM), 0.5 μLreverse primer (10 μM), 0.2 μLprobe (10 μM), 0.2 μLreverse transcriptase, 0.5 μLTaq DNA polymerase, 5 μL5X reaction buffer, and 13.1 μLsterilized water. All these components were fully mixed in a PCR tube and added to the microfluidic chip by a pipette, then PCR amplification was performed. The process of the amplification included Step One: reverse transcription at 50°C for 15 minutes; Step Two: denaturation at 95°C for 3 minutes; and Step Three: 40 cycles at 95°C for 15 seconds and at 55°C for 45 seconds.
Real-time PCR conditions. DNA or cDNA was amplified by a real-time PCR using Power SYBR Green PCR Master Mix Kit in OneStep ABI instrument. Each reaction had a total volume of 25 μL, including 12.5 μL SYBR Green master mix, 200 nmol of each forward and reverse primers, 5 μL cDNA plus 7.1 μL ddH2O. The cycling conditions included an initial denaturation step of 10 minutes at 94°C, followed by 40 cycles of 15 seconds at 95°C, one minute at 55°C and one minute at 60°C. Fluorescent detection was at the end of each cycle. Melting curve analysis program was used for identification of specific PCR products.
Chip design and preparation. There were 8 reaction chambers with a volume of 2 μL each on the PCR microfluidic chip. The PCR microfluidic chips were made of silicon wafers of 500 μm thick with both sides polished and heat-resistant glass substrates of 500 μm thick by Shenzhen Shineway Technology Corporation. The PCR reaction chambers and channels on the silicon wafers were formed by dry etching using a semiconductor photolithography process. Then, sixteen holes were drilled on the silicon substrate which served as the inlets and outlets of PCR solutions, respectively. Finally, the silicon wafers were bonded to the glass substrates and the two-layer wafers were cut by a dicing saw to form independent PCR detection chips.
a standardized semiconductor fabrication process was utilized to ensure the yield rate and uniformity in quality of the chips. And the automated fabrication process could significantly reduce the cost of each single inspection by expanding the size of the silicon wafers.
The amplification detection system of the PCR microfluidic chip. A portable rapid microfluidic PCR system (SWA-01, Shenzhen Shineway Technology Corporation) was used to perform the real-time PCR amplification of the samples (Fig. 1). The dimension was 330mm(L)×320mm(W)×160mm(H)and the weight was round 3 kg, suggesting it had a small footprint. Used in conjunction with the specially supplied reagents and the microfluidic chips, the SWA-01 PCR instrument is applicable to rapid real-time fluorescence detection of PCR in the fields of animal quarantine, food safety and public health and safety. The portable rapid microfluidic PCR instrument was mainly composed of a control system (with a touch screen graphic user interface), a power supply system, a photoelectric system, a temperature control system [35], an outer jacket part, a microfluidic chip, and a software module. The instrument had the following advantages for on-site detection as follows: portable design, low weight, rapid thermal cycling and real time detection.