Table_1_Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.docx

BackgroundClassical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care.
MethodsA loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated.
ResultsThree primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles.
ConclusionThe results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

研究背景：经典猪瘟病毒（Classical swine fever virus, CSFV）仍是动物健康领域最重要的病原之一。当前的病原检测流程需先提取病毒RNA，再进行逆转录实时荧光定量PCR（RT-qPCR）检测。目前亟需开发新型技术以提升现场即时诊断能力。
研究方法：本研究开发了环介导等温扩增（loop-mediated isothermal amplification, LAMP）PCR技术，引物设计覆盖所有已报道的CSFV基因型。分别采用荧光检测与比色检测两种方式对该扩增反应进行验证，并以金标准检测方法作为对照。实验测试了3种流行CSFV基因型的病毒毒株以及感染动物的样本。同时还检测了其他多种病原，以评估LAMP方法的特异性。除实验室常规RNA提取方法外，本研究还评估了一种可适配现场条件的简易RNA释放加热法。
研究结果：本研究共设计了3组引物，其中1组引物表现最优。该引物组可在60℃~68℃的宽幅扩增温度区间内保持最佳检测性能，且能够有效检测上述3种基因型的CSFV RNA。该检测方法可高效检出2种不同基因型野外毒株感染动物的样本，且通过比色与荧光两种检测方式均可实现多种样本基质的检测。LAMP检测方法对所有受试非相关病原均呈阴性反应，包括瘟病毒属（Pestivirus）病原。仅在针对新型意大利绵羊瘟病毒（ovine Italy Pestivirus, OVPV，即Pestivirus italiaense）的检测中，荧光与比色LAMP均出现了疑似阳性结果；该病毒已被证实可与多种CSFV检测技术发生交叉反应，但仅当病毒载量高于10000个病毒粒子时，才能通过该疑似阳性结果检出OVPV。
研究结论：本研究结果表明，LAMP技术可作为现有CSFV分子诊断技术的重要补充手段。由于该技术仅需少量设备即可适配现场使用，且可采用微创样本进行检测，因此可应用于偏远地区。将新型与传统诊断技术联合使用，可作为支持猪瘟防控工作的有效备选方案。