Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h.
Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h.
复制标题
DOI：
10.1016/j.mimet.2013.06.015
复制
发表时间：
2013-09
影响因子：
2.2
通讯作者：
Rees, Catherine E. D.
中科院分区：
文献类型：
--
作者：
Swift, Benjamin M. C.;Denton, Emily J.;Mahendran, Sophie A.;Huxley, Jonathan N.;Rees, Catherine E. D.
本研究旨在建立一种快速检测禽分枝杆菌活菌的方法。临床血液样品中的副结核菌素（MAP）。使用优化的肽介导的磁性分离（PMMS）回收掺入市售血液中的MAP细胞，并使用基于噬菌体的方法进行检测，并使用MAP特征序列（IS 900）的巢式PCR扩增确认检测到的细胞的身份。检测限确定为每ml血液10个MAP细胞，并用于检测临床牛血液样品中存在的MAP。使用PMMS-噬菌体方法，当从全血或从分离的血沉棕黄层检测MAP时没有差异。通过PMMS-噬菌体在牛奶-ELISA阳性的动物（15只动物）中检测到MAP，并且在来自经认证的无约翰氏病牛群（5只动物）的血液样品中未检测到MAP。在来自具有可变乳汁ELISA状态的动物的一个畜群（10头动物）的一组样品中，除一种情况外，PMMS噬菌体结果与阳性乳汁ELISA结果一致。这些结果表明PMMS-噬菌体方法可以检测自然感染血液中存在的MAP。总测定时间为48小时，与基于PCR的检测试验不同，仅检测活细胞。一种快速检测血液中MAP的方法可以进一步了解约翰氏病动物的播散性感染。使用磁分离优化血液中有效的MAP细胞捕获发现在加标血液中的检测限为101 pfu ml− 1优化PCR以检测来自仅一个噬斑的特征MAP DNA序列我们在48小时内成功地在自然感染的动物中检测到活MAP
The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease. Optimisation of efficient MAP cell capture in blood using magnetic separation Found a limit of detection of 101 pfu ml− 1 in spiked blood Optimised a PCR to detect signature MAP DNA sequences from just one plaque We successfully detected viable MAP in naturally infected animals within 48 h
登录
查看更多内容
影响因子：
4.4
作者：
Foddai, Antonio;Elliott, Christopher T.;Grant, Irene R.
通讯作者：
Grant, Irene R.
影响因子：
4
作者：
Grant, IR;Kirk, RB;Rowe, MT
通讯作者：
Rowe, MT
影响因子：
1.1
作者：
Gwozdz, JM;Thompson, KG;Manktelow, BW
通讯作者：
Manktelow, BW
影响因子：
3.5
作者：
Raizman, E. A.;Fetrow, J. P.;Wells, S. J.
通讯作者：
Wells, S. J.
影响因子：
3.3
作者：
Reddacliff, LA;Vadali, A;Whittington, RJ
通讯作者：
Whittington, RJ