环介导等温核酸扩增技术快速检测产毒亚历山大藻

DNA环介导等温扩增(LAMP)方法是一种新型的核酸扩增技术,该方法是在等温的条件下进行的,具有反应时间短、灵敏度高、特异性强的特点.本文以产麻痹性贝毒(PSP)的亚历山大藻为研究对象,采用简易法提取DNA模板,设计特异性LAMP引物,利用LAMP技术进行产毒藻种的快速检测,同时,着重对LAMP技术与PCR技术在检测微小亚历山大藻细胞的灵敏度方面做了比较.向LAMP终产物中加入SYBR Green I 染料后可直接用肉眼观察结果而不需要通过凝胶电泳来观察.结果表明,LAMP技术在恒温65℃,1h内就可以检测到产毒藻种;LAMP技术检测微小亚历山大藻的最低检测限为200个/ml,而PCR技术的最低检测限为1000个/ml,LAMP扩增方法比PCR扩增方法的程序简单、反应时间短、灵敏度高;LAMP技术不需要精密的温度循环装置,有恒温加热设备就可以满足检测条件,可用于野外检测.     

恒温实时荧光法检测副溶血性弧菌

为了更加准确、快速地检测进出口食品中的副溶血性弧菌,针对副溶血性弧菌的tlh基因设计环介导等温扩增(loop-mediatedisothermal amplification,LAMP)引物,结合ESEQuant tube scanner来对扩增结果进行实时检测。结果显示:该引物扩增效率高、特异性强、灵敏度高(1-10 CFU/反应,约为荧光PCR的100倍)。126株副溶血性弧菌,恒温荧光法检测出126株,荧光PCR的方法检测出121株。恒温实时荧光检测仪与荧光PCR仪类似,均可以对检测结果进行实时监测和熔解曲线分析,表明该恒温实时荧光检测仪可广泛应用于基础实验室的科学研究或经济发展相对落后区域的临床初步诊断。     

快速检测淋球菌porA和16S rRNA基因的环介导等温扩增技术的建立

目的建立快速检测淋球菌porA和16S rRNA基因的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术,探讨其用于淋球菌感染早期诊断的可行性。方法利用Primer-ExplorerV3软件分别针对淋球菌porA及16S rRNA基因设计6条引物(2条内引物FIP、BIP,2条外引物F3、B3,2条环引物LF、LB),以淋球菌标准菌株基因组DNA为模板,进行LAMP扩增,同时,以外引物F3、B3为PCR引物,进行PCR扩增,并比较2种扩增方法的灵敏度和特异性。结果LAMP法与PCR法灵敏度相同,可检测到10个拷贝的目的基因;其针对淋球菌porA和16S rRNA基因的引物对脑膜炎奈瑟菌、肺炎链球菌和大肠埃希菌的DNA均不能扩增。结论已成功建立了检测淋球菌porA和16S rRNA基因的LAMP技术,为淋球菌的快速检测提供了新的手段,有望成为淋球菌常规检测的简便方法。     

食品中大肠杆菌0157: H7的几种检测方法的比较

目的 评估平板分离培养法、免疫磁珠分离(IMS)法、VIDAS全自动酶标免疫测试系统、BAX全自动病原菌检测系统及环介导等温扩增(LAMP)技术在食品中检验肠出血型大肠埃希菌0157:H7的特异性、敏感性.方法 使用平板分离培养法、免疫磁珠分离法、VIDAS法、BAX法及LAMP法对人工制备的染菌猪肉样本进行检测,并对这几种方法进行比较.结果 BAX法和LAMP法的检出率最高,分别是89.1％和85.9％,免疫磁珠法和VIDAS法检出率次之,分别是75.0％和78.1％,传统分离培养法为43.8％.结论 BAX法和LAMP法具有快速、高效、特异性好、敏感性高的特点,可快速筛选食品中可能存在的肠出血型大肠埃希菌0157∶H7.     

环介导等温扩增技术检测食品中铜绿假单胞菌

建立食品中铜绿假单胞菌环介导等温扩增(LAMP)检测方法。利用铜绿假单胞菌外毒素A(PEA)基因序列,设计3对铜绿假单胞菌LAMP检测特异性引物,用36株铜绿假单胞菌,14株近源菌验证方法的特异性。建立的LAMP方法特异性好,灵敏度达到2.2 cfu/100 g~3.5 cfu/100 g。建立的食品中铜绿假单胞菌LAMP检测方法特异性好,灵敏度高,适用于的检测食品中的铜绿假单胞菌。     

环介导恒温扩增法鉴定牛羊肉中的搀杂肉

建立了一个鉴定牛羊肉中搀杂杂动物肉的环介导恒温扩增检测方法。确定了一套可在牛羊肉中特异并灵敏地检测出搀杂肉成分的引物对,以动物细胞色素b基因组为模板可在恒温63℃恒温特异性扩增出猪等基因片段而无其他扩增片段影响。可检测牛肉中0.01%～2%的猪肉成分。经与PCR方法比对,结果表明,该方法可有效用于实际生鲜肉或加工肉制品样本的鉴定。     

Propidium monoazide for viable Salmonella enterica detection by PCR and LAMP assays in comparison to RNA-based RT-PCR,RT-LAMP,and culture-based assays

Rapid and sensitive detection of live/infectious foodborne pathogens is urgently needed in order to prevent outbreaks and food recalls. This study aimed to (1) evaluate the incorporation of propidium monoazide (PMA) into PCR or LAMP assays to selectively detect viable Salmonella Enteritidis following sublethal heat or UV treatment, and autoclave sterilization; and (2) compare the detection of PMA-PCR and PMA-LAMP to DNA-based PCR and LAMP (without PMA), RNA-based RT-PCR and RT-LAMP, and culture-based methods. Nucleic acids (DNA or RNA) from 1-mL S. Enteritidis samples were used for PCR, RT-PCR, LAMP, and RT-LAMP assays. Serially diluted samples were plated on Xylose Lysine Tergitol-4 agar for cultural enumeration. Comparable detection of overnight cultured S. Enteritidis was obtained by PMA-PCR, PCR, and RT-PCR, though 1 to 2 log less sensitive than cultural assays. PMA-LAMP and RT-LAMP showed similar detection of overnight cultures, being 1 to 2 log less sensitive than the LAMP assay, and ∼4 log less than culture-based detection. Autoclaved S. Enteritidis did not test positive by RNA-based methods or PMA-PCR, but PMA-LAMP showed detection of 1 log CFU/mL. PMA-PCR and RT-PCR showed comparable detection of sublethal heat-treated cells to cultural assays, while PMA-LAMP showed 1 to 2 log less detection. Our results suggest that PMA-PCR and PMA-LAMP assays are not suitable for selective viable cell detection after UV treatment. While PMA-LAMP assay needs optimization, PMA-PCR shows promise for live/viable S. Enteritidis detection. PMA-PCR shows potential for routine testing in the food industry with results within 1-day, albeit depending on the inactivation method employed.     

Rapid and Sensitive Identification of the Herbal Tea Ingredient Taraxacum formosanum Using Loop-Mediated Isothermal Amplification

Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.     

The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants

Bacterial pathogens and toxicants, parasites as well as mycotoxin producing fungi are the major biotic factors influencing the safety of food. Moreover, viral infections and prions may be present as quasi biotic challenging factors. A vast array of culture dependent analytical methods and protocols for food safety testing has been developed during the past decades. Presently, protocols involving molecular biological techniques such as PCR-based nucleic acid amplification and hybridization have become available for many of the known pathogens with their major advantages being rapidness, high sensitivity and specificity. However, this type of assays is still quite labor- and cost intensive and mostly cannot be operated directly in the field. Recently, loop-mediated isothermal amplification (LAMP) of DNA has emerged as an alternative to the use of PCR-based methods not only in food safety testing but also in a wide array of application. Its advantages over PCR-based techniques are even shorter reaction time, no need for specific equipment, high sensitivity and specificity as well as comparably low susceptibility to inhibitors present in sample materials which enables detection of the pathogens in sample materials even without time consuming sample preparation. The present article presents a critical review of the application of LAMP-based methods and their usefulness in detecting and identifying food borne bacterial pathogens and toxicants as well as mycotoxin producing food borne fungi as compared to other methods. Moreover does it elaborate on new developments in the design and automation of LAMP-based assays and their implications for the future developments of food testing.     

食品沙门氏菌环介导等温扩增技术应用研究进展

沙门氏菌引起的人畜共患疾病每年引发大量财产损失,快速简易的检验方法对于食品沙门氏菌的检验至关重要。环介导等温扩增（Loop-mediated isothermal amplification, LAMP）作为PCR的有力替代工具,经历多年发展已在等温扩增领域显示出明显优势,在沙门氏菌检测应用方面也日渐成熟。本文对近五年来LAMP技术在沙门氏菌检测中的应用和LAMP技术检测平台进行了分析汇总。在沙门氏菌LAMP技术的特异性方面,对现有靶标基因的有效性和新靶标基因的有效性进行了探讨,对不同血清型的特异性检测和多重LAMP的应用进行了总结,分析其优点与不足之处,以此说明探针法、多重检测,微流控技术和即时检测（Point-of-Care Testing, POCT）是沙门氏菌LAMP技术的未来发展方向,为LAMP技术更好地应用于沙门氏菌检测提供参考依据,对于提高食品中沙门氏菌的检验能力具有重要意义。