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

背景：由于鸡毒支原体(MG)和滑膜支原体(MS)，英国鸡禽业损失了数百万鸡蛋和数百吨肉类。这些病原体导致呼吸道、生殖和肌肉骨骼感染，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，还没有用于抗体或抗原检测的笔侧快速诊断横向流动试验。目的：建立和评价用于检测家禽临床样品中MG和MS抗体或抗原的横向流动(LF)试验。假设和目标(48个月博士学位)：1-12个月：MG/MS侧向血流(LF)抗体(Ab)测试的进展。MG(Mgc2，GAPA和PLPA)(1，2)和MS(VlhA)(3)细胞粘附素蛋白重组蛋白的合成、纯化和定量这将通过重组和在大肠杆菌中表达来实现(4)。超声波MG/MS(5)也将包括在内。LF抗体测试开发将包括胶体金(CG)纳米颗粒的制备、CG与抗血清、硝酸纤维素膜、样品和连接垫的结合(6)。6-18个月：建立MG/MS LF抗原(AT)试验，以鉴定临床标本中MG/MS的表面蛋白。将制备和纯化一组MG/MS表面蛋白单抗(MAb)。随后，生物素标记的单抗将使用生物素间隔物和胶体金偶联物(7)。如上所述，将使用LF测试条，并对所涉及的部件进行优化。将建立抗体/At检测的敏感度和特异度。12-24月：在单一LF试条中同时检测MG/MS的优化。在单次检测中成功检测和优化MG和MS后，将对程序进行修改，以单条检测MG/MS抗体或抗原。25-30日：活体实验感染MG/MS：将通过眼鼻接种SPF鸡(8只)。每周采集血液和口咽-后鼻孔裂拭子，直到比赛结束后6周。月31-36：使用实验鸡的样本(上图)对LF进行内部验证。血/血清和拭子(上图)将分别用于LF抗体和AT试验。测试设备将得到优化。月37-42：使用临床样本对LF进行验证。来自Aviagen鸡群的样本在农场和他们的实验室进行了测试。结果与市售的MG/MS ELISA和PCRS进行交叉比较。42-48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、VIVA和出版物。

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.