Development and evaluation of lateral-flow test for rapid detection of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry clinical samples

背景资料：由于鸡毒支原体（MG）和滑液支原体（MS），英国家禽业损失了数百万枚鸡蛋和数百吨肉类。这些病原体会引起呼吸道、生殖道和肌肉骨骼感染，导致疾病、药物费用增加和产蛋量下降。到目前为止，对于MG和MS，没有笔侧快速诊断侧流检测，无论是抗体或抗原detection.Aims：侧流（LF）检测的发展和评价检测MG和MS抗体或抗原在家禽临床样品。假设和目的（48个月PhD）：第1-12个月：MG/MS侧流（LF）抗体（Ab）试验的开发。针对MG（mgc 2、GapA和PlpA）（1，2）和MS（vlhA）（3）的细胞粘附素蛋白的重组蛋白的合成、纯化和定量。这将通过质粒重组和在E. coli（4）。还将包括超声MG/MS（5）。LF Ab检测开发将包括胶体金（CG）纳米颗粒的制备、CG与抗血清的结合、硝酸纤维素膜、样品和结合垫（6）。第6-18个月：开发MG/MS LF抗原（At）检测，以鉴定临床样本中MG/MS的表面蛋白。将制备并纯化一组MG/MS表面蛋白单克隆抗体（mAb）（见上文）。随后，生物素化的mAb将使用生物素-间隔物和胶体金缀合物（7）。将使用如上所述的LF试纸，并对相关组件进行优化。将建立Ab/At检测的灵敏度和特异性。第12-24个月：优化单LF试纸条检测MG/MS。在单次检测中成功检测和优化MG和MS后（如上所述），将修改程序，以在单个试剂条中检测MG/MS抗体或抗原。第25-30个月：MG/MS的体内实验性感染：将通过眼鼻接种SPF鸡（8）。攻毒后6周内，每周采集一次血液和口咽-后鼻孔裂拭子。第31-36个月：使用来自实验鸡的样品（上文）进行LF的内部验证。血液/血清和拭子（上述）将分别用于LF Ab和At检测。将优化测试设备。第37-42个月：使用临床样本验证LF。安伟捷鸡群的样本在农场和实验室进行了测试。结果与商业MG/MS ELISA和PCR交叉比较。第42-48个月：完成工作和论文写作。完成实验室工作和数据分析，并撰写论文。最后3个月：论文提交，论文发表。

Background: The UK chicken poultry industry loses millions of eggs and hundreds of tons of meat due to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). These pathogens cause respiratory, reproductive and musculoskeletal infections, resulting in illnesses, increased medication costs and drops in egg production. To date, for MG and MS, there are no pen-side rapid diagnostic lateral flow tests available, for either antibody or antigen detection.Aims: Development and evaluation of lateral flow (LF) test for detection of MG and MS antibodies or antigens in poultry clinical samples. Hypotheses and objectives (48 months PhD):Months 1-12: Development of MG/MS lateral flow (LF) antibody (Ab) test. Synthesis, purification and quantification of recombinant protein against cytadhesin proteins of MG (mgc2, GapA and PlpA)(1, 2) and MS (vlhA) (3). This will be achieved by plasmid recombination and expression in E. coli (4). Sonicated MG/MS (5) will also be included. LF Ab test development will include preparation of colloidal gold (CG) nanoparticles, conjugation of the CG with the antisera, nitrocellulose membrane, sample and conjugation pads (6). Month 6-18: Development of MG/MS LF antigen (At) test to identify surface proteins of MG/MS in clinical samples. A panel of MG/MS surface protein monoclonal antibodies (mAb) (above) will be prepared and purified. Subsequently, the mAb biotinylated will use a biotin-spacer and colloidal-gold conjugated (7). LF test strip, as above, will be used and go through optimisation of components involved. Sensitivity and specificity of Ab/At detection will be established.Month 12-24: Optimisation for detection of both MG/MS in single LF strip test. Following successful detection and optimisation of MG and MS in single test (above), the procedures will be modified to test MG/MS antibodies or antigens in a single strip. Month 25-30: In vivo experimental infection of MG/MS: SPF birds will be inoculated via oculonasal (8). Blood and oropharyngeal-choanal cleft swabs will be collected weekly up to 6 weeks post challenge. Month 31-36: Internal validation of LF using samples (above) from experimental chickens. The blood/sera and swabs (above) will be used in the LF Ab and At test respectively. Test devices will be optimised. Month 37-42: Validation of the LF using clinical samples. Samples from Aviagen flocks tested in the farms and in their laboratory. Results cross-compared against commercial MG/MS ELISAs and PCRs. Month 42-48: Completion of work and thesis writing. Complete laboratory work and data analysis, also writing thesis. Last 3 months: thesis submission, viva and publications.