Spiroplasma DNA Detection Kit (qPCR), BP-QN299
Detects Spiroplasma contamination by probe-based qPCR.
Product overview
This reagent kit uses the PCR-fluorescence probe method to design specific primers and probes for the conserved regions of Spiroplasma 16S rRNA sequence, which can specifically amplify Spiroplasma DNA with the characteristics of easy operation, rapid detection, strong specificity and high sensitivity.
Features and applications
- Simple operation: The workflow from sample extraction through completion of testing takes only 1.5 hours.
- Sensitivity: The limit of detection reached 10 CFU/mL.
- Precision: Intermediate precision (Ct value) CV ≤5%.
- Specificity: No cross-reactivity with common cell lines and structurally related microorganisms.
- The kit is used for qualitative detection of Spiroplasma DNA contamination in cells, cell and gene therapy products, and other biological products.
Components
| Components | Specification/Notes |
|---|---|
| Spi qPCR MIX | 1.0 mL x 1 tube |
| Spi Positive Control | 1.5 mL x 2 tubes |
| Spi Negative Control | 1.5 mL x 2 tubes |
| Spi Internal Control | 1.0 mL x 1 tube |
| Instructions | 1 copy |
Performance data
qPCR Sensitivity Verification
The pharmacopoeia-specified Spiroplasma validation strain at 10 CFU/mL was extracted and tested 24 times. Results are shown in Table 1.
Table 1. Summary of LOD Data
| Strain | Concentration (CFU/mL) | Detection rate |
|---|---|---|
| Spiroplasma citri | 10 | 24/24 |
At 10 CFU/mL, the kit detected the standard Spiroplasma strain in all 24 test replicates.
Cross-Reactivity Results
Cross-reactivity was evaluated using DNA from commonly used cell lines, bacteria, and Mycoplasma. No cross-reactivity was observed with Hi5, Sf9, Clostridium sporogenes, SP2/0, C6, Escherichia coli, Pseudomonas aeruginosa, Mycoplasma orale, Mycoplasma pneumoniae, Micrococcus luteus, Cutibacterium acnes, Streptococcus pyogenes, HeLa, and Vero DNA. Results are shown below.
Table 2. Cross-Reactivity Results
| No. | Sample | Replicate 1 | Replicate 2 |
|---|---|---|---|
| 1 | Hi5 | Negative | Negative |
| 2 | Sf9 | Negative | Negative |
| 3 | C. sporogenes | Negative | Negative |
| 4 | SP2/0 | Negative | Negative |
| 5 | C6 | Negative | Negative |
| 6 | E. coli | Negative | Negative |
| 7 | P. aeruginosa | Negative | Negative |
| 8 | M. orale | Negative | Negative |
| 9 | M. pneumoniae | Negative | Negative |
| 10 | M. luteus | Negative | Negative |
| 11 | C. acnes | Negative | Negative |
| 12 | S. pyogenes | Negative | Negative |
| 13 | HeLa | Negative | Negative |
| 14 | Vero | Negative | Negative |
Conclusion:
None of the panel-listed genomic-DNA materials (Hi5, Sf9, C. sporogenes, SP2/0, C6, E. coli, P. aeruginosa, M. orale, M. pneumoniae, M. luteus, C. acnes, S. pyogenes, HeLa, and Vero) exhibited cross-reactivity with the Spiroplasma DNA Detection Kit.
Bioinformatics Analysis
Sequence-alignment of kit-specific primers and probe against the NCBI database indicated coverage of more than 40 reported Spiroplasma species.
Precision
Within-Run Precision
In-house reference materials Z1, Z2, and Z3 were tested in 10 replicates each. Precision CV was within 5% at every concentration.
Intermediate Precision
Intermediate precision was evaluated across operators, days, instruments, and reagent lots using in-house reference materials Z1, Z2, and Z3, with five replicates per material. Precision CV was within 5% at every concentration.
Total Imprecision
Data across operators, days, instruments, and reagent lots were pooled. Precision CV was within 5% at every concentration.
Performance data shown here were obtained with the reference materials, extraction workflows, instruments and acceptance criteria used in BIORI’s product-specific validation study. Users should verify performance in their own sample matrix and workflow.
Shipping and storage
| Condition | Recommendation |
|---|---|
| Storage | ≤-18°C, protected from light. Shelf life: 24 months. Avoid more than 7 freeze-thaw cycles. |
| Shipping | Ship frozen on dry ice or with validated frozen cold-chain packaging that maintains the specified frozen storage condition. |
| Handling | Protect from light and avoid unnecessary freeze-thaw cycles where applicable. |
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