Biofunctionalized Magnetic Nanoparticles with Multiplex Touchdown PCR for Simultaneous and Rapid Detection/Identification of Campylobacter jejuni and Campylobacter coli
Biofunctionalized Magnetic Nanoparticles with Multiplex Touchdown PCR for Simultaneous and Rapid Detection/Identification of Campylobacter jejuni and Campylobacter coli
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DOI：
10.1155/2022/5104187
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发表时间：
2022-10-12
影响因子：
--
通讯作者：
Tuitemwong，Pravate
中科院分区：
文献类型：
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作者：
Wenbap，Pattarapong;Rattanarojpong，Triwit;Tuitemwong，Pravate
简单、准确、快速地检测食源性致病菌对公众健康至关重要。免疫磁分离多重触地聚合酶链式反应(IMS-Mplex TD-PCR)同时检测和鉴别OFC方法的建立JejuniandC.本文报道了结肠菌属。将抗多表位抗原(MEA)的多克隆抗体(Pab)偶联到铁磁性纳米颗粒(FMN)上，制备抗MEA的FMN。最佳载药量为26.7μg/mg FMN，平均粒径为72±9 nm，PAb结合率为83%。抗MEA FMN(20μg)在纯培养条件下以3.5 4×10 2个集落形成单位(CFU)/m L的浓度捕获可培养的空肠上皮细胞，而较高的浓度(40和60μg)降低了回收率。扫描电子显微镜(SEM)分析表明，抗MEA FMN附着在靶细菌上，形成聚集的细胞和椭圆形的磁性纳米颗粒。随后的多重TD-PCR方法同时检测和鉴别C。JejuniandC.Coliat 104CFU/m L混合培养，每个菌种103cfu/m L。此外，IMS多重TD-PCR方法在添加鸡胸肉样品中的检出限为104CFU/g。两种方法的特异性均为100%。JejuniandC.在没有弯曲杆菌的对照样品中，未检测到任何一种扩增产物。该方法能够检测和区分C。JejuniandC.该方法简便、准确、快速，无需浓缩步骤即可检测到4 h，是一种快速、无菌检测鸡肉制品中空肠弯曲菌素的方法。
The simple, accurate, and rapid detection of foodborne pathogens is essential for public health. Development of an immunomagnetic separation (IMS) multiplex touchdown PCR (IMS–multiplex TD–PCR) assay for simultaneous detection and distinguishing ofC. jejuniandC. coliis reported herein. Polyclonal antibody (pAb) against multiepitope antigen (MEA) was conjugated to ferromagnetic nanoparticles (FMNs) to produce anti‐MEA FMNs. Optimal anti‐MEA FMNs loading yielded 26.7μg of immunoglobulin G (IgG) molecules per mg of FMNs with an average size of 72 ± 9 nm, corresponding to an 83% rate of pAb conjugation. Anti‐MEA FMNs (20μg) for IMS captured culturable C.jejunicells at 3.54 × 102colony‐forming unit (CFU)/mL in pure culture, while higher amounts (40 and 60μg) reduced the recovery. The scanning electron microscope (SEM) analysis revealed the attachment of anti‐MEA FMNs to target bacteria, forming aggregated cells and magnetic nanoparticles in ellipse‐like shapes. The subsequent multiplex TD–PCR assay simultaneously detected and distinguishedC. jejuniandC. coliat 104CFU/mL in mixed culture and at 103CFU/mL for each individual species. Furthermore, the limit of detection (LOD) of the IMS–multiplex TD–PCR assay was 104CFU/g in spiked chicken breast samples. Specificity was 100% for bothC. jejuniandC. colias none of the amplicons were detected in control samples whereCampylobacterwas absent. This assay is able to detect and distinguishC. jejuniandC. colisimultaneously and is simple, accurate, and rapid with a time to result of 4 h without an enrichment step, making it a promising approach for rapid and culture‐free detection ofCampylobacterin chicken products.