Identification by subtractive hybridization of sequences specific for Salmonella enterica serovar Enteritidis
Identification by subtractive hybridization of sequences specific for Salmonella enterica serovar Enteritidis
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DOI：
10.1128/aem.67.11.4984-4991.2001
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发表时间：
2001-11-01
影响因子：
4.4
通讯作者：
Andersen, GL
中科院分区：
文献类型：
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作者：
Agron, PG;Walker, RL;Andersen, GL
肠道沙门氏菌是食物中毒的主要原因，可以通过未彻底煮熟的完整鸡蛋传播给人类。鸡的感染是无症状的;因此，简单、灵敏和特异的检测方法对于限制人类接触至关重要。抑制性消减杂交用于分离存在于沙门氏菌血清型肠道沙门氏菌中但在家禽环境中发现的其他细菌中不存在的DNA限制性片段。在聚合酶链反应中使用候选区域的寡核苷酸引物来检测73个非肠道菌群。肠杆菌分离株包括34个不同血清型，包括都柏林和鸡白痢，肠杆菌的两个非常近的亲戚。一个沙门氏菌差异片段（称为Sdf I）的引物对清楚地区分了所有其他血清型肠杆菌，而其他两个引物对只确定了一些非肠杆菌菌株。这些引物对也可用于检测临床和环境中肠道沙门氏菌血清型分离株的多样性。此外，未检出常见于肠道沙门氏菌血清型的5个细菌属。通过用降解剪切的染色体DNA而不是完整的环状质粒DNA的核酸外切酶处理总DNA，表明Sdf I位于染色体上。Sdf I引物用于筛选沙门氏菌肠道血清型基因组文库，并定义了独特的4，060-bp区域。这些结果提供了一个快速，灵敏，高特异性的检测系统，沙门氏菌血清型肠道沙门氏菌的基础上，并提供序列信息，可能是相关的独特的特点，这种血清型。
Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.