Description

[Product Specifications] BPMPro100 100 Reactions/kit

[Product Description] Mycoplasma: Currently known as the smallest prokaryotic organism, statistics show that approximately 15-35% of cells are contaminated with mycoplasma. Mycoplasma contamination can alter the structure and function of host cells, leading to unstable experimental results. Regular mycoplasma testing of cultured cells is necessary.

This kit employs dual-color fluorescence qPCR (TaqMan probe method) for mycoplasma DNA detection. Test samples can include cell culture supernatant, cell precipitate, or other biological products. The kit features the following characteristics: Sensitive: Combined with the recommended nucleic acid extraction protocol, detection sensitivity reaches 10 CFU/ml. Reliable: Internal control nucleic acid added during extraction ensures complete quality control. Stable: Closed-tube operation prevents cross-contamination. Convenient: Simple operation, no electrophoresis steps required.

[Shelf Life] 6 months under specified storage conditions. After removal from -20°C, can be stored at 4°C for 1 week protected from light.

[Kit Components] Table 1 Kit Components

Notes:

1. Positive control contains mycoplasma DNA and internal control DNA.
2. Internal control contains internal control DNA. Please read this manual completely before experimentation and strictly follow sterile operation protocols!

[Operating Procedures] 1. Sample Preparation (1) Standard sample preparation protocol: Please use an efficient contamination-free DNA extraction kit to extract sample DNA. The entire process must follow sterile operation protocols. Our currently recommended extraction kits are listed in Appendix 3. Directly take 20 µL of test sample DNA as template for qPCR reaction. (2) Pre-treatment negative sample preparation: Use RNase Free Water as sample, add internal control, and perform nucleic acid extraction.

2. qPCR Reaction Mix Preparation (1) Calculate required reaction wells based on sample number, with 3 replicates per sample. Reaction wells = 1 positive PCR control + 1 negative PCR control + 1 pre-treatment negative control + sample number × 3 (2) Calculate total qPCR Mix needed based on reaction wells (include 1 well for pipetting loss): Mix = (reaction wells + 1) × 10 μl (3) Thaw reagents at room temperature and prepare qPCR Mix according to table below:

Table 2 qPCR Mix Preparation

3. Sample Loading (1) Vortex mix qPCR Mix, briefly centrifuge to collect liquid at bottom. (2) Dispense 10 μL qPCR Mix into each reaction tube. (3) Add 15 μl mineral oil to reaction tubes containing qPCR Mix. (4) Add samples to reaction tubes and briefly centrifuge, loading example as follows: ** Table 3 Loading Example**

[Note]: Total volume in each reaction well is 30μL. qPCR negative PCR template uses 20 ul RNase Free Water. PCR instrument settings using ABI 7500 as example, relative quantification mode, select TaqMan probe method:

Notes: 1. This program uses two-stage amplification. If fluorescence quantitative PCR instrument can collect fluorescence in two stages, judge results by normal Ct value; if fluorescence can only be collected in final step, add 15 Ct values to original data. ** 2. ROX settings use 7500 as example, but exceptions exist, such as StepOne.**

[Threshold Settings] Using ABI 7500 as example, select log method for amplification curves. If two-stage fluorescence collection is not possible, set baseline to 1-2 cycles; if possible, set baseline to 3-15 cycles. Log method amplification curves recommended for stable and reliable results. (1) Internal reference (HEX/VIC) threshold setting: Set internal reference amplification curve threshold using positive control, set internal reference Ct value to 28±2. (2) Mycoplasma (FAM) threshold setting: Set mycoplasma amplification curve threshold using positive control, set mycoplasma (FAM) Ct value to 28±2.

[Experimental Quality Control] If positive control tube mycoplasma (FAM) Ct value >30, but positive control tube and pre-treatment negative control tube internal reference (HEX) Ct values are normal, indicates positive mycoplasma control template degradation. If sample pre-treatment negative internal reference (HEX/VIC) Ct value >30, positive control tube internal reference (HEX) Ct value >30, indicates internal reference DNA degradation or PCR system amplification efficiency decrease.

[Result Interpretation] Based on mycoplasma (FAM) Ct value and internal standard (VIC) Ct value, specific criteria as follows:

Each test sample requires 3 replicates; if 2 replicates are positive among 3 replicates, interpret as positive. Sample pre-treatment negative control recommended for quality control of entire extraction process.

Note: For abnormal detection results:

1. Sample pre-treatment negative: High internal reference Ct value indicates low extraction efficiency; mycoplasma detection channel Ct value <37.5 may indicate contamination during pre-treatment extraction process.
2. qPCR negative: Mycoplasma and internal reference detection channel Ct value <37.5 indicates operational contamination.
3. Difference of 1±1 Ct value between sample pre-treatment negative control and PCR positive control internal reference is normal.

[PCR Process Precautions] Due to PCR reaction sensitivity, note following points during operation to prevent DNA contamination:

1. After kit opening, store positive control and internal control separately from reagents Biowing® MyqPCR Reaction Mix1, Biowing®MyPrimer&Probe Mix2, RNase Free Water, and mineral oil. 2. Briefly centrifuge each component before use and carefully open. When using, gently invert ten times to mix, avoid bubbles, and briefly centrifuge before use.
2. Nucleic acid extraction and qPCR processes must comply with sterile operation protocols.
3. Recommend using transfer box to transport prepared Mix from Mix preparation clean bench to sample loading clean bench.
4. Recommend arranging positive control on qPCR plate away from test samples and negative control.
5. Recommend performing qPCR preparation and dispensing at one sterile workstation, sample loading at another sterile workstation.
6. Recommend using different pipettes for positive control versus test samples and negative control, and use filter tips for loading.
7. Load in order: negative first, then samples, positive last. Use dedicated pipette for positive control. Recommend loading positive control at dedicated sterile workstation.
8. Repeatedly press firmly when sealing qPCR plate or closing tubes.
9. Brief low-speed centrifugation before amplification to collect liquid at bottom
10. After closing reaction tube caps or applying optical film, avoid marking on caps or film, or repeatedly rubbing with plate scraper, to prevent interference with fluorescence signal reading.
11. This product is for research use only.