Test Label: Biotin labeled primer / FAM probeTest Format: AmplifyRP® XRTInternal Control: Yes - Exogenous
AmplifyRP XRT for Foc TR4 is a rapid DNA amplification and detection platform designed for testing banana root, rhizome, stem, pseudostem, and leaf for Fusarium oxysporum f. sp. cubense tropical race 4 (Fusarium odoratissimum), the causal agent of Panama Disease. This kit includes lyophilized reaction pellets containing the necessary reagents to amplify Foc TR4 DNA and an exogenous DNA control at a single operating temperature (42 °C).
AmplifyRP XRT is a real-time isothermal DNA amplification and detection system that rapidly amplifies small portions of DNA and offers unrivaled detection capabilities in an easy-to-use testing format. It offers comparable sensitivity and specificity to published PCR methods while eliminating laborious and costly nucleic acid extractions.
The test can be performed virtually anywhere using the battery operated AmpliFire fluorometer. Assay parameters are loaded via barcode and results are automatically displayed as ( + ) or ( - ).
Prior molecular diagnostic experience is not required to perform AmplifyRP XRT tests.
NOTE: This assay requires a fluorometer to work properly.
Limit of Detection (LOD): 25 pg of genomic DNA; 40 copies of DNA fragments per reaction
Specificity:
- Inclusive: Designed to detect all Fusarium oxysporum f. sp. cubense tropical race 4 strains. Strains detected include: NRRL 36114 (VCG 01213), NRRL 54006 (Foc-TR4 reference strain Focll5) and 5 isolates from VCG 01213/16.
- Exclusive:
- Does not detect: Amplicon DNA fragments from homologous genes of Foc-R1, R2, R3 and STR4. Fungal species Colletotrichum gloeosporioides, Fusarium monoliforme (NRRL 13911), Fusarium pallidoroseum (NRRL 20425), Fusarium solani (NRRL 22587), Fusarium subglutinans (NRRL 25734), Rhizoctonia solani (NRRL 66082, DSM 843, DSM 852, DSM 22843, DSM 22847, DSM 62716, DSM 63002), Fusarium oxysporum f.sp. cubense STR4, Fusarium oxysporum f.sp. cubense R1, Phytophthora citrophthora, Phytophthora heveae, Phytophthora palmivora, Phytophthora kernoviae, Phytophthora cryptogea, Phytophthora ramorum, Pythium aphanidermatum, Pythium irregulare, Pythium ultimum,
- Predicted not to detect via insilico data: Botryosphaeia dothoidea, Colletotrichum gleosporioides (including musae), Alternaria alternata, Cladosporium musae, Mychosphaerella fifienses, Mycorrhizae spp, Xanthomonas spp, or Ralstonia spp.
- No reaction to healthy Banana root, rhizome, stem, pseudostem and leaf.
- Technical Information
- Includes
Total Assay Time: Less than 30 minutes when used with the AmpliFire as a real-time assay.
Includes:
- XRT reaction pellets for Foc TR4 (48)
- Pre-filled 100 µL PD1 Pellet diluent tubes (48)
- AMP1 extraction buffer (55 mL)
- Sample bag, mesh (50)
- User Guide
NOTE: This assay requires a fluorometer to work properly.
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二、限制性内切酶消化样品DNA
三、凝胶电泳分离消化产物
四、如果靶序列>5kb,在0.25M的HCl中进行震荡脱嘌呤10min,ddH2O洗一次
五、用变性液震荡处理30min,ddH2O洗一次
六、中和液震荡处理30min
七、裁取合适大小的尼龙膜或硝酸纤维素膜进行转膜操作,可用真空转膜仪或者搭滤纸桥,需要20×SSC
八、制备探针,可用地高辛标记(以地高辛为例)
九、将膜放入杂交瓶,42°C预杂交30min
十、倒掉预杂交液,加入杂交液,适当的温度进行杂交4h至过夜
十一、洗膜,先用2×SSC+0.1%SDS,20-25°C洗2×5min,再用0.5×SSC+0.1%SDS洗2×15min。然后用washing buffer洗1-5min,接着用blocking solution洗30min后,用antibody solution洗30min,再用washing buffer洗2×15min,再用detection buffer洗2-5min后,取出膜,放于保鲜膜上,在结合有DNA的一面滴加CSPD ready-to-use后,立刻盖上保鲜膜,让CSPD ready-to-use均匀的布满膜表面,室温放置5min后,37°C温育10min以上
十二、放射自显影,可用成像系统信号累积模式显影或用X-ray显影
应该注意以下2个方面
1 用玻璃棒将气泡赶净 有气泡的位置不导电 蛋白就转不上
2 半干式转膜仪要保证一定量的转移缓冲液,如果转膜一个小时以上,缓冲液要多一些
我用Bio-rda转膜仪,转膜2分钟就停止,然后机器上显示“noloaddetected“。各位知道这是为什么吗??
第一次出现这种情况,同样的条件,之前一直没有问题。

