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添加扩增内标的沙门氏菌PCR检测方法

, PP. 54-59

Keywords: 沙门氏菌,PCR检测,扩增内标,假阴性

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Abstract:

通过构建人工扩增内标,建立可以有效指示沙门氏菌检测过程可能出现假阴性情况的PCR检测方法。本研究基于沙门氏菌invA基因设计特异性引物,复合法构建扩增内标,建立PCR检测体系。特异性引物LW,对33株沙门氏菌和6株非沙门氏菌标准株进行检测,结果显示,所有沙门氏菌均扩增出385bp的目标片段,非沙门氏菌则只能扩增出484bp的扩增内标片段,特异性良好。灵敏度实验表明,该检测体系的灵敏度可达6.35fg/μL。人工污染实验表明,起始染菌量为3.2CFU/25mL时,仅需8h增菌培养便可检出。大量食品样品检测证明,该检测体系确实可以有效的避免PCR检测过程出现的假阴性,提高检测准确性。

References

[1]  杨保伟, 张秀丽, 曲东, 等. 2007-2008陕西部分零售畜禽沙门氏菌血清型和基因型[J]. 微生物学报, 2010, 50(50):654-660.
[2]  Moreira MAB, Luvizotto MCR, Garcia JF, et al. Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs [J]. Veterinary Parasitology, 2007, 145(3):245-252.
[3]  González-Escalona N, Brown EW, Zhang GD. Development and evaluation of a multiplex real-time PCR(qPCR)assay targeting ttrRSBCA locus and invA gene for accurate detection of Salmonella spp. in fresh produce and eggs[J]. Food Research International, 2012, 48(1):202-208.
[4]  Malorny B, Hoorfar J, Bunge C, et al. Multicenter validation of the analytical accuracy of Salmonella PCR:towards an international standard[J]. Applied and Environmental Microbiology, 2003, 69(1):290-296.
[5]  Malorny B, Tassios PT, R?dstr?m P, et al. Standardization of diagnostic PCR for the detection of foodborne pathogens [J]. International Journal of Food Microbiology, 2003, 83(1):39-48.
[6]  Fricker M, Messelh?uβer U, Busch U, et al. Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks[J].Applied and Environmental Microbiology, 2007, 73(6):1892-1898.
[7]  史贤明, 索标食. 源性致病菌分子检测技术研究进展[J]. 农产品质量与安全, 2010, 3:36-41.
[8]  索标, 汪月霞, 艾志录, 等.食源性致病菌多重分子生物学检测技术研究进展[J]. 微生物学杂志, 2010, 30(6):71-75.
[9]  Burggraf S, Olgemoller B. Simple technique for internal control of real-time amplification assays [J]. Clinical Chemistry, 2004, 50(5):819-825.
[10]  Hoorfar J, Malorny B, Abdulmawjood A, et al. Practical considera-tionsin design of internal amplification controlsfor diagnostic PCR assays [J]. Journal of ClinicalMicrobiology, 2004, 42(5):1863-1868.
[11]  Maaroufi Y, de Bruyne M, Duchateau V, et al. Development of amu-liple internal control for clinical diagnostic real-time amplification assays [J]. Fems Immunology and Medical Microbiology, 2006, 48(2):183-191.
[12]  卢强, 陈贵连, 林万明. PCR扩增invA基因特异性检测沙门氏菌[J]. 中国兽医学报, 1994, 14(3):251-256.
[13]  Hadjinicolaou A, Demetriou V, Emmanuel M, et al. Molecular bea-con-based real-time PCR detection of primary isolates of Salmonella typhimurium and Salmonella enteritidis in environmental and clin-ical samples[J]. BMC Microbiology, 2009, 9(1):97.
[14]  Siebert PD, Larrick JW. PCR MIMICS:competitive DNA fragments for use as internal standards in quantitative PCR[J]. Biotechniques, 1993, 14(2):244-249.
[15]  刘斌, 史贤明. 扩增内标在沙门氏菌 PCR 检测方法中的应用[J]. 微生物学通报, 2006, 33(2):156-161.
[16]  何晓华, 史贤明. 扩增内标及其在食源性致病菌PCR检测中的应用[J]. 微生物学报, 2010, 50(2):141-147.
[17]  Rahn K, De Grandis SA, Clarke RC, et al. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J]. Molecular and Cellular Probes, 1992, 6(4):271-279.
[18]  González-Escalona N, Fey A, H?fle M G, et al. Quantitative reverse transcription polymerase chain reaction analysis of Vibrio cholerae cells entering the viable but non-culturable state and starvation in response to cold shock[J]. Environmental Microbiology, 2006, 8(4):658-666.
[19]  Nordstrom JL, Vickery MCL, Blackstone GM, et al. Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemol-yticus bacteria in oysters[J]. Applied and Environmental Micro-biology, 2007, 73(18):5840-5847.
[20]  Diez-Valcarce M, Kova? K, Cook N, et al. Construction and analytical application of internal amplification controls(IAC)for detection of food supply chain-relevant viruses by real-time PCR-based assays[J]. Food Analytical Methods, 2011, 4(3):437-445.

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