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Survey of the distribution of staphylococcal enterotoxin C (SEC) subtypes and SEG, SEH, SEI gene in staphylococcal food-borne outbreaks and application of the internal transcribed spacer sequence (ITS) for Salmonella detection
16S-23S internal transcribed spacer
在本研究中建立一個快速檢測腸炎沙門氏菌的方法，我們從插入序列中針對S. Enteritidis 設計一組具特異性之PCR檢測引子IS1/IS2。經檢測其特異性之後，完全不受非沙門氏菌之干擾，僅受S. Berta 及 S. Gallinarum兩株菌之干擾，而此兩菌株未曾在台灣地區出現過，故此引子組可利用為分子檢測工具，及一般檢測腸炎沙門氏菌的參考。另外，我們由PCR檢測腸炎沙門菌相關的文獻中合成四組引子組，經評估其檢測特異性，發現Soumet等人(1996)發展的S1/S4引子組特異性最佳。
本研究從內轉錄區和23S rRNA基因中，設計一組能擴增ITS區域的通用引子(ITSA1及ITSB1)。利用所設計之一組通用引子，成功的將25種血清型(40株) 沙門氏菌和8株非沙門氏菌部分ITS片段增幅出來。接著進行DNA序列定序工作，利用基因序列分析軟體，進行基因多條序列比對後找出具有變異的區域，進而設計出可檢測所有血清型沙門氏菌之專一性PCR引子組(ITSF/ITSR)。此專一性PCR引子組經特異性測試，不同血清型之沙門氏菌及其臨床菌株皆會產生312 bp之PCR擴增產物，非沙門氏菌的革蘭氏陽性及陰性菌株則沒有產生相同之PCR產物。以煮沸法破壞沙門氏菌細胞進行靈敏度試驗，純菌均可達N×100 (N=1~9) CFU / per assay之目標菌數。ITSF/ITSR直接檢測全脂鮮乳及雞肉食品樣品之沙門氏菌的靈敏度試驗，均可達103 CFU/ml或gram 之目標菌數。若將N×100 (N=1~9) CFU / per gram or ml 之S. Enteritidis (ATCC 13076)菌液接種於食品樣品中，取出均質液經1﹪buffered peptone water、37℃ 8小時的預培養，靈敏度可達N×100 (N = 1~9) CFU/ml或gram之目標菌數。|
ABSTRACT Staphylococcal enterotoxins (SEs), first identified in 1959, are a group of extracellular proteins produced by some strains of Staphylococcus aureus. While Vibrio parahaemoliticus (860 cases) has been the leading cause of foodborne diseases, Staphylococcus aureus and related enterotoxins are the second most frequently encountered cause of foodborne diseases (247 cases) in Taiwan. However, the aetiology of a high percentage of the of staphylococcal foodborne poisoning (SFP) cases remains unclear. SEs are classified as members of the pyrogenic toxin (PT) superantigen family because of their biological activities and structural relatedness. They are unlike the other members of the PT family, the SEs have the unique ability to induce staphylococcal food poisoning, a common form of gastroenteritis. Twelve serologically distinct enterotoxins, i.e. enterotoxin A (SEA), SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEK, SEL and SEM, have been identified. The SEC serotype is heterogenous and contains several antigenic and sequence molecular variants, designated SEC1, SEC2, SEC3, SECbovine, and SECovine. The classification is based on the minor antigenic differences and the animal host with which they are infected. Amino acid sequences of the SEC1, SEC2 and SEC3 and the nucleotide sequence of these SEC genes have been identified. However, additional SEC variants, other than SEC1, SEC2 and SEC3, do exist. Commercially available immunoassay kits, such as the staphylococcal enterotoxins reverse phase latex agglutination (SET-RPLA) kit, and the staphylococcal enterotoxin ELISA (SET-EIA) kit, only allow the detection of classical SEs (i.e. SEA, SEB, SEC, SED and SEE) but not the specific individual subtypes of SEC. DNA probes and the polymerase chain reaction (PCR) primers are the two methods available for DNA detection. These probes and primers are specific for the detection of various staphylococcal enterotoxin genes. For SEC subtypes, there is only one report regarding the method for detection and differentiation of SEC subtypes. Due to the high homology of the amino acid sequence in SEC1, SEC2 and SEC3, the method for differentiation of SEC subtypes is not yet commercially available. Based on the nucleotide sequences for these enterotoxins, we have designed SEC subtype specific primers for the detection of these SEC genes individually. We have used those SEC subtype specific primers to investigate the distribution of SEC1, SEC2 and SEC3 S. aureus strains in staphylococcal food poisoning outbreaks occurred in the central Taiwan in 1995-2000. Despite the high homology (97%) in their gene sequences, we designed a new set of PCR primers with which we were able to differentiate each specific SEC subtypes. We have used these primers for the differentiation of 39 strains of SEC S. aureus isolated from patients suffered with food poisoning between 1995-2000 in the central Taiwan. In the recent years, the existence of new types of SEs (SEG, SEH, SEI, SEJ, SEK, SEL, and SEM) has also been reported. However, the relationship between these new SEs and food poisoning is not fully understood at the present time. It is estimated that about 95% of staphylococcal food poisoning outbreaks are caused by SEA to SEE. The remaining 5% of outbreaks may therefore be associated with the other newly identified SEs. To clarify the role played by these newly identified SEs in food poisoning, the development of reliable methods of detection of SE proteins is essential. However, characterization of these SEs has been hindered because of the low levels of production, difficulties associated with purification, the lack of simple and practical methods for their detection. The only reliable assay for the unidentified SEs is the monkey feeding test. However, this test cannot differentiate serologically different SEs. Among the techniques used to identify toxin genotypes, PCR have been reported to be very successful and reliable. Our laboratory previously designed specific primers for the successful and reliable detection of SEs, ETA, ETB, and TSST-1. Purification of an unidentified SE will allow the development of a specific and sensitive PCR assay for rapid and reliable detection. Therefore, we developed a PCR procedure that will rapidly assess whether staphylococcal isolates harbor seg, seh, and sei, encoding the SEs. A total of 116 clinical isolates of S. aureus obtained from patients suffered with food poisoning and 55 clinical isolates other of S. aureus negative to the classical enterotoxins (SEA® SEE) detection were selected for testing. All isolates were identified with the BAM method and SET-RPLA system. PCR assays were performed for the classical enterotoxins (SEA® SEE) gene sequences. In this report, novel PCR primers specific for the detection of SEG, SEH and SEI genes, ie, seg, seh and sei, were designed and used for the assay of 55 human isolates of S. aureus negative to the classical enterotoxins (SEA® SEE) detection. Human salmonellosis occurs in a variety of forms including gastroenteritis, organ focal infection, or systemic febrile infection. Salmonellae are widely distributed in the environment and are harboring in both wild and domestic animals. A number of salmonella serotypes may be transferred to poultry from various sources, such as feedstuffs, breeding flocks, rodents, and wild birds. The conventional methods for detection and identification of Salmonella spp. include the preculture process, selective enrichment and biotypes identification and the serotyping process for O, Vi and H antigens. According to the O, Vi and H sero grouping, strains of Salmonella spp. can then be identified. Since these procedures are laborious, the development of rapid identification methods is important. The purpose of this study is to develop a PCR system that can be used for the detection of Salmonella spp. and S. Enteritidis. Detection sensitivity and reliability for these systems and the conditions for application of such systems on food and clinical samples will also be investigated. S. Enteritidis is one of the most common Salmonella species that may cause food-borne disease and salmonellosis. The Centers for Disease Control and Prevention (USA) reported a fivefold increase in S. Enteritidis isolation rates in the northeastern and central Atlantic states between 1976 and 1985. In this study, we would design the PCR system specific for S. Enteritidis detection. From the insertion sequence (Accession no. Z83734), PCR primers specific for S. Enteritidis detection is designed IS1/IS2. Only S. Berta and S. Gallinarum may generate false results. The results indicated that the primers could be used as a molecular tool for future field survey of S. Enteritidis both in foods and in clinical samples. The feasibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was also investigated by direct sequencing of polymerase chain reaction-amplified DNA from all ITS regions in a collection of 40 strains of 25 different serotypes of S. enterica, and by sequencing individual ITS region from a single strain. The sequence contained internal transcribed spacer fragments of 485—490 bases in length. Based on the sequences in the amplified ITS regions, we designed a pair of PCR primers (ITSF and ITSR) which could be used for the specific detection of Salmonella spp. with all serovars. The uniqueness of these primer sequences was confirmed. Molecular size of the PCR product is 312 bp and all the non-Salmonella strains tested generated negative PCR results. The sensitivity for boiled (97℃, 30 min) S. Typhimurium whole bacteria cell was N ×100 (N = 1~9) cells per assay. When this PCR primer pair was used for the detection of Salmonella cells contaminated in cow milk and raw chicken meat, the detection limit was 103 cfu per g or ml of the food samples. For food samples spiked with N ×100 (N = 1~9) CFU per g or ml S. Enteritidis (ATCC 13076) cells, homogenised, inoculated into 1﹪buffered peptone water and grown at 37℃ for 8 hours, the detection of S. Enteritidis using the PCR was successful.
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