Q：40612 一步法快速支原體檢測試劑盒（防污染版）的檢測原理？

A：本試劑盒中加入了UDG 酶，以便去除含 dU 的PCR 產(chǎn)物氣溶膠污染，在極大程度上抑制假陽性反應，提高檢測的準確度。而且加入的 UDG 酶在室溫下起作用，且對溫度感、易于滅活，不影響實驗結果。若細胞培養(yǎng)物被支原體污染，支原體 DNA 的保守序列會被大量、快速地擴增，使反應液由藍紫色變成天藍色，結果肉眼可辨，無需電泳。

Q：一步法快速支原體檢測試劑盒使用方法（防污染版）？

A：本試劑盒使用更為簡便，只需吸取 1μL 疑似支原體污染的細胞培養(yǎng)上清加入反應體系中，先將樣品在 25~37 ℃放置 5 min，再在水浴鍋或 PCR 儀中，恒溫 63 °C 孵育 60 min。觀察反應液由藍紫色變成天藍色，結果肉眼可辨，無需電泳。

Q：用 40612 一步法支原體檢測試劑盒檢測 DMSO、乙醇等有機溶劑中是否存在支原體污染，發(fā)現(xiàn)實驗組均是天藍色，是不是 DMSO、乙醇等時機中存在支原體污染？

A：支原體試劑盒在乙醇、DMSO 有機溶劑，以及強酸強堿環(huán)境中，不穩(wěn)定易變性，導致顏色變色為天藍色。這款試劑盒只能用于細胞檢測，不可用于有機溶劑、強酸強堿環(huán)境下檢測。

Q：我檢測的是這個里面的液體，加入之后PCR管里的顏色立馬變成了天藍色？是什么原因呢？檢測胰酶-EDTA，加入樣品之后，直接變色了？

A：40612原理：Mg2++HNB=藍紫色，隨著PCR增加，焦磷酸不斷增多，焦磷酸與Mg2+結合，HNB游離，顯示天藍色。這個試劑里面有磷酸二氫鈉可能是這個離子與Mg2+結合而立馬產(chǎn)生化學反應導致的，EDTA可以螯合鈣鎂離子，從而導致反應加速發(fā)生變色反應。

Q：陽性對照的濃度時多少？

A：10的5次方copies/ul。

Q：陽性對照里的是支原體嗎？

A：不是的，是線性化的片段。

[1] Shi N, Yang Q, Zhang H, et al. Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids. Nat Biomed Eng. 2022;6(2):195-206. doi:10.1038/s41551-021-00774-1(IF:25.671)
[2] Zhang S, Yu F, Che A, et al. Neuroendocrine Regulation of Stress-Induced T Cell Dysfunction during Lung Cancer Immunosurveillance via the Kisspeptin/GPR54 Signaling Pathway. Adv Sci (Weinh). 2022;9(13):e2104132. doi:10.1002/advs.202104132(IF:16.806)
[3] Li Y, Xue B, Zhang M, et al. Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency. Genome Biol. 2021;22(1):206. Published 2021 Jul 12. doi:10.1186/s13059-021-02424-w(IF:13.583)
[4] Wu L, Xu Y, Zhao H, et al. FcγRIIB potentiates differentiation of myeloid-derived suppressor cells to mediate tumor immunoescape. Theranostics. 2022;12(2):842-858. Published 2022 Jan 1. doi:10.7150/thno.66575(IF:11.556)
[5] Tan B, Shi X, Zhang J, et al. Inhibition of Rspo-Lgr4 Facilitates Checkpoint Blockade Therapy by Switching Macrophage Polarization. Cancer Res. 2018;78(17):4929-4942. doi:10.1158/0008-5472.CAN-18-0152(IF:9.130)
[6] Yan G, Zhao H, Zhang Q, et al. A RIPK3-PGE₂ Circuit Mediates Myeloid-Derived Suppressor Cell-Potentiated Colorectal Carcinogenesis. Cancer Res. 2018;78(19):5586-5599. doi:10.1158/0008-5472.CAN-17-3962(IF:9.130)
[7] Gu Z, Shi C, Li J, et al. Palbociclib-based high-throughput combination drug screening identifies synergistic therapeutic options in HPV-negative head and neck squamous cell carcinoma. BMC Med. 2022;20(1):175. Published 2022 May 12. doi:10.1186/s12916-022-02373-6(IF:8.775)
[8] Wu L, Zhang X, Zheng L, et al. RIPK3 Orchestrates Fatty Acid Metabolism in Tumor-Associated Macrophages and Hepatocarcinogenesis. Cancer Immunol Res. 2020;8(5):710-721. doi:10.1158/2326-6066.CIR-19-0261(IF:8.728)
[9] Qin J, Zhang X, Tan B, et al. Blocking P2X7-Mediated Macrophage Polarization Overcomes Treatment Resistance in Lung Cancer. Cancer Immunol Res. 2020;8(11):1426-1439. doi:10.1158/2326-6066.CIR-20-0123(IF:8.728)
[10] Cao M, Huang W, Chen Y, et al. Chronic restraint stress promotes the mobilization and recruitment of myeloid-derived suppressor cells through β-adrenergic-activated CXCL5-CXCR2-Erk signaling cascades. Int J Cancer. 2021;149(2):460-472. doi:10.1002/ijc.33552(IF:7.396)
[11] Yao R, Alkhawtani AYF, Chen R, Luan J, Xu M. Rapid and efficient in vivo angiogenesis directed by electro-assisted bioprinting of alginate/collagen microspheres with human umbilical vein endothelial cell coating layer [published correction appears in Int J Bioprint. 2020 Sep 17;6(4):309]. Int J Bioprint. 2019;5(2.1):194. Published 2019 Jun 24. doi:10.18063/ijb.v5i2.1.194(IF:6.638)
[12] Liu L, Deng Y, Zheng Z, et al. Hsp90 Inhibitor STA9090 Sensitizes Hepatocellular Carcinoma to Hyperthermia-Induced DNA Damage by Suppressing DNA-PKcs Protein Stability and mRNA Transcription. Mol Cancer Ther. 2021;20(10):1880-1892. doi:10.1158/1535-7163.MCT-21-0215(IF:6.261)
[13] Wu S, Yang X, Tang W, et al. Chemotherapeutic Risk lncRNA-PVT1 SNP Sensitizes Metastatic Colorectal Cancer to FOLFOX Regimen. Front Oncol. 2022;12:808889. Published 2022 Mar 31. doi:10.3389/fonc.2022.808889(IF:6.244)
[14] Wu Q, Xuan YF, Su AL, Bao XB, Miao ZH, Wang YQ. TNKS inhibitors potentiate proliferative inhibition of BET inhibitors via reducing β-Catenin in colorectal cancer cells. Am J Cancer Res. 2022;12(3):1069-1087. Published 2022 Mar 15. (IF:6.166)
[15] Wu L, Zhao KQ, Wang W, et al. Nuclear receptor coactivator 6 promotes HTR-8/SVneo cell invasion and migration by activating NF-κB-mediated MMP9 transcription. Cell Prolif. 2020;53(9):e12876. doi:10.1111/cpr.12876(IF:5.753)
[16] Wei Z, Wang Y, Peng J, et al. CircRFWD3 promotes HNSCC metastasis by modulating miR-27a/b/PPARγ signaling. Cell Death Discov. 2022;8(1):285. Published 2022 Jun 11. doi:10.1038/s41420-022-01066-6(IF:5.241)
[17] Chen W, Weng Z, Xie Z, et al. Sequencing of methylase-accessible regions in integral circular extrachromosomal DNA reveals differences in chromatin structure. Epigenetics Chromatin. 2021;14(1):40. Published 2021 Aug 23. doi:10.1186/s13072-021-00416-5(IF:4.954)
[18] Wu Z, Zheng M, Zhang Y, et al. Hsa_circ_0043278 functions as competitive endogenous RNA to enhance glioblastoma multiforme progression by sponging miR-638. Aging (Albany NY). 2020;12(21):21114-21128. doi:10.18632/aging.103603(IF:4.831)
[19] Wang L, Zhou Y, Cao C, et al. The exon 12-containing LHX6 isoforms promote cervical cancer cell proliferation by regulating the MAPK signaling pathway [published online ahead of print, 2022 Apr 5]. Cancer Med. 2022;10.1002/cam4.4734. doi:10.1002/cam4.4734(IF:4.452)
[20] Zhang Q, Yan G, Lei J, et al. The SP1-12LOX axis promotes chemoresistance and metastasis of ovarian cancer. Mol Med. 2020;26(1):39. Published 2020 May 6. doi:10.1186/s10020-020-00174-2(IF:4.096)
[21] Hu J, Wu Q, Wang Z, et al. Inhibition of CACNA1H attenuates doxorubicin-induced acute cardiotoxicity by affecting endoplasmic reticulum stress. Biomed Pharmacother. 2019;120:109475. doi:10.1016/j.biopha.2019.109475(IF:3.743)
[22] Wang T, Lin F, Sun X, et al. HOXB8 enhances the proliferation and metastasis of colorectal cancer cells by promoting EMT via STAT3 activation. Cancer Cell Int. 2019;19:3. Published 2019 Jan 3. doi:10.1186/s12935-018-0717-6(IF:3.439)
[23] Meng LL, Wang JL, Xu SP, et al. Low serum gastrin associated with ER⁺ breast cancer development via inactivation of CCKBR/ERK/P65 signaling. BMC Cancer. 2018;18(1):824. Published 2018 Aug 16. doi:10.1186/s12885-018-4717-7(IF:3.288)
[24] Ma J, Liu X, Liu P, et al. Identification of a new p53 responsive element in the promoter region of anillin. Int J Mol Med. 2020;45(5):1563-1570. doi:10.3892/ijmm.2020.4527(IF:3.098)
[25] Chen L, Chen L, Wan L, et al. Matrine improves skeletal muscle atrophy by inhibiting E3 ubiquitin ligases and activating the Akt/mTOR/FoxO3α signaling pathway in C2C12 myotubes and mice. Oncol Rep. 2019;42(2):479-494. doi:10.3892/or.2019.7205(IF:3.041)
[26] Men XM, Xu ZW, Tao X, Deng B, Qi KK. FNDC5 expression closely correlates with muscle fiber types in porcine longissimus dorsi muscle and regulates myosin heavy chains (MyHCs) mRNA expression in C2C12 cells. PeerJ. 2021;9:e11065. Published 2021 Apr 19. doi:10.7717/peerj.11065(IF:2.984)
[27] Zhao YQ, Wu T, Wang LF, et al. Targeting MUC1-C reverses the cisplatin resistance of esophageal squamous cell carcinoma in vitro and in vivo. Transl Cancer Res. 2021;10(2):645-655. doi:10.21037/tcr-20-2495(IF:1.241)