Three new methods applying a novel approach for rapid and simple detection of specific bacteria, based on plaque formation as the end point of the phage lytic cycle, are described. Different procedures were designed to ensure that the resulting plaques were derived only from infected target bacteria ("infectious centers"). (i) A pair of amber mutants that cannot form plaques at concentrations lower than their reversion rate underwent complementation in the tested bacteria; the number of plaques formed was proportional to the concentration of the bacteria that were coinfected by these phage mutants. (ii) UV-irradiated phages were recovered by photoreactivation and/or SOS repair mediated by target bacteria and plated on a recA uvrA bacterial lawn in the dark to avoid recovery of noninfecting phages. (iii) Pairs of temperature-sensitive mutants were allowed to coinfect their target bacteria at the permissive temperature, followed by incubation of the plates at the restrictive temperature to avoid phage infection of the host cells. This method allowed the omission of centrifuging and washing the infected cells. Only phages that recovered by recombination or complementation were able to form plaques. The detection limit was 1 to 10 living Salmonella or Escherichia coli O157 cells after 3 to 5 h. The antibiotic susceptibility of the target bacteria could also be determined in each of these procedures by preincubating the target bacteria with antibiotic prior to phage infection. Bacteria sensitive to the antibiotic lost the ability to form infectious centers.

译文

:描述了三种新方法,这些新方法应用了一种新颖的方法来快速,简单地检测特定细菌,该方法以噬菌斑形成为噬菌体裂解周期的终点。设计了不同的程序以确保所得的噬菌斑仅来自受感染的目标细菌(“感染中心”)。 (i)在测试细菌中对一对不能以低于其回复速率的浓度形成噬菌斑的琥珀突变体进行互补;噬菌斑形成的数量与被这些噬菌体突变体共同感染的细菌的浓度成正比。 (ii)通过目标细菌介导的光活化和/或SOS修复来回收紫外线照射的噬菌体,并在黑暗中接种在recA uvrA细菌草坪上,以避免非感染性噬菌体的恢复。 (iii)允许成对的温度敏感突变体在允许的温度下共同感染它们的靶细菌,然后在极限温度下孵育平板以避免噬菌体感染宿主细胞。这种方法可以省去离心和洗涤感染细胞的步骤。只有通过重组或互补而恢复的噬菌体才能形成噬菌斑。 3至5小时后,检出限为1至10个活沙门氏菌或大肠杆菌O157细胞。还可以通过在噬菌体感染之前将目标细菌与抗生素一起预孵育,在每种方法中确定目标细菌的抗生素敏感性。对抗生素敏感的细菌丧失了形成感染中心的能力。

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