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

描述了基于噬菌斑形成作为噬菌体裂解周期终点的三种新方法,该方法应用了一种新方法来快速,简单地检测特定细菌。设计了不同的程序以确保所产生的斑块仅来自受感染的目标细菌 (“感染中心”)。(i) 一对在低于其还原率的浓度下无法形成斑块的琥珀突变体在被测细菌中进行了互补; 形成的斑块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,并在黑暗中将其铺在reco uvrA细菌草坪上,以避免恢复非感染性噬菌体。(iii) 允许成对的温度敏感突变体在允许的温度下共同感染其目标细菌,然后在限制性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清洗被感染的细胞。只有通过重组或互补回收的噬菌体才能形成斑块。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。在每个程序中,也可以通过在噬菌体感染之前将目标细菌与抗生素预孵育来确定目标细菌的抗生素敏感性。对抗生素敏感的细菌失去了形成感染中心的能力。

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