Identification of Mycobacterium Species by DNA Microarray Chip Method

[Narrator] This research focuses on molecular diagnostics in clinical microbiology. Compared with traditional detection methods, the DNA microarray chip method for identifying mycobacterium species is highly sensitive, specific, and rapid, which can provide important assistance for the early diagnosis of clinical patients. Currently, rapid identification methods for nontuberculous mycobacteria are limited in many grassroots laboratories, which directly impact patient clinical diagnosis and treatment. To begin, set the temperature of the constant temperature water bath to 50 degrees Celsius and preheat it. To prepare chips, add 200 microliters of distilled water to the bottom of the gene microarray chip hybridization box. Place the bracket between the two positioning columns inside the box. Carefully place the chip with the front side up on the bracket and cover it with the cover slip so that it's four bosses face downward, ensuring the end of the cover slip aligns with the label at the end of the chip. Next, prepare 200-microliter centrifuge tubes, or eight well strips according to the number of samples and label them properly. Heat the hybridization buffer from the kit at 50 degrees Celsius until it is completely melted. After thorough mixing, centrifuge it quickly in a micro centrifuge. Aliquot nine microliters of hybridization buffer into each prepared tube or strip. Add six microliters of the corresponding PCR product to each tube, making a final volume of 15 microliters per reaction mixture. Heat the hybridization reaction mixture to 95 degrees Celsius for five minutes to denature it using a PCR instrument or water bath. After the denaturation treatment, immediately immerse the mixture in an ice water mixture and incubate for three minutes. Remove the hybridization reaction mixture from the ice bath and mix it by pipetting up and down twice. When no white flocculent precipitate remains, add 13.5 microliters of the reaction mixture through the loading hole of the cover slip. Quickly cover and seal the hybridization box and record the chip number, microarray position, and sample number. Now, place the sealed hybridization boxes horizontally into the constant temperature water bath that has been preheated to 50 degrees Celsius. After placing all the boxes inside, start the timer for 120 minutes. After the hybridization reaction is completed, take out the hybridization boxes horizontally, disassemble them, and retrieve the chips. Immediately place the chips on the slide rack inside a container filled with Chip Washing Solution I that has been equilibrated to room temperature and wash them at 80 to 100 RPM on a constant temperature shaker for three minutes at room temperature. Wash the chips again with Chip Washing Solution II on the shaker set to 80 to 100 RPM for three minutes at room temperature. Now, place the chips in a centrifuge and spin at 100 g for five minutes at room temperature. Turn on the scanner. And then open the corresponding software. Click the Laser Control button to allow it to preheat for 10 minutes. Input sample-related information into the software. After the scanner finishes preheating, click the Eject button. Place the dried chip steadily on the small bracket piece. Push the chip gently and horizontally into the scanner compartment and click the Load button. Now, input the chip number into the software and click to select the detection area microarray 1 to 4. Click Select Sample to choose the corresponding samples for each microarray. Then click Start Detection to begin chip scanning. The scanning results will be displayed on the screen and automatically saved. Use the data query page to conduct data queries and printing operations. After completing all operations, turn off the laser, exit the software, and finally, turn off the scanner. The validity of the experimental results is confirmed by quality control with the positive control yielding a detection result of mycobacterium tuberculosis complex and the negative control yielding no mycobacterium. Interpretation of test samples begins with cases where all probes, including the one with the highest signal value, are negative, resulting in a report of no mycobacterium. In samples where only the mycobacterium tuberculosis complex probe shows a strong signal, the result is identified as positive for that species group. If the probe corresponding to mycobacterium avium is the only one with a strong signal, the sample is identified as positive for mycobacterium avium. If only the internal control probe is positive and all others are negative, the result is reported as indeterminate, indicating the presence of a mycobacterium species not covered by the detection kit.