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1.
We evaluated the TaqMan PCR Salmonella amplification/detection kit (PE Applied Biosystems) for rapid detection of Salmonella from a variety of meat samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The detection sensitivity of the kit, using 2 kinds of DNA extraction protocols, was compared with that obtained with 4 protocols of official culture methods. A total of 98 meat samples (16 raw beef, 31 pork and 51 chicken) were tested. The results of the TaqMan PCR method and the combined results of the 4 cultural protocols showed excellent agreement. However, no single culture protocol showed optimal recovery of Salmonella comparable to the PCR method. These results suggest that the TaqMan PCR method is a reliable and rapid method useful for detecting Salmonella in meat products.  相似文献   

2.
Detecting internal Salmonella Enteritidis (SE) contamination in eggs is essential for protecting public health. Pooling together > or = 10 eggs for sampling allows many eggs to be screened for contamination, but such pools must be incubated (usually at 25 to 37 degrees C) to permit small numbers of SE to multiply before further testing. The present study determined whether incubating egg contents pools at an elevated temperature (42 degrees C) could increase the rate of multiplication of a phage type 14b strain of SE sufficiently to support the detection of contamination by a rapid lateral flow immunodiffusion method within a single day. Pools of 10 eggs were contaminated with approximately 10 CFU of SE, supplemented with concentrated broth enrichment medium, and incubated at either 37 or 42 degrees C. Incubation of contaminated egg pools at 42 degrees C resulted in significantly higher SE levels after 6, 8, 10, and 12 h. However, incubation at 42 degrees C could only generate a mean log SE concentration of 4.21 CFU/ml within a single working day (8 h), inadequate to support efficient detection by most rapid assays. Detection of SE contamination in egg pools by a rapid lateral flow immunodiffusion test was not achieved at a high frequency until 12 h of incubation at 42 degrees C.  相似文献   

3.
An assay was developed for the specific detection of Salmonella Enteritidis in eggs with the use of an application of the fluorogenic 5' nuclease assay (TaqMan). In this assay, a segment of the gene sefA specific to Salmonella group D strains such as Salmonella Enteritidis was used. The amplification of the target gene products was monitored in real-time by incorporating a fluorescent dye-labeled gene-specific probe in the PCR reaction. This method correctly detected and distinguished Salmonella Enteritidis from nearly 50 of non-group D Salmonella and other non-Salmonella strains. Detection of the sefA gene was linear for DNA extracted from approximately 10(2) to 10(9) CFU/ml in phosphate-buffered saline and 10(3) to 10(8) CFU/ml in raw egg. In two trials, when applied to detection of Salmonella Enteritidis in homogenized egg pools and compared with conventional culture methods, the newly developed PCR method yielded a 100% correlation with results obtained by a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the culture method. The sensitivity of this assay was approximately less than 1 CFU/600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low concentrations of Salmonella Enteritidis in egg samples.  相似文献   

4.
Salmonella is the leading cause of foodborne illnesses in the United States, and Salmonella Enteritidis (SE) is the second most frequently isolated Salmonella serovar. Egg products are most often associated with outbreaks of SE infection. To prevent SE contamination of eggs, many producers are implementing flock inspections for SE at their facilities. A rapid and simple method for detecting SE in poultry environmental samples is critical for effective control of SE. In this study, the Reveal test for SE was compared with the conventional U.S. Food and Drug Administration (FDA) culture method for detecting SE in naturally contaminated environmental samples. The efficacy of two enrichment media, tetrathionate broth (TT) and Rappaport-Vassiliadis medium (RV), and three selective plating media, brilliant green agar with novobiocin (BGN), xylose lysine tergitol 4 agar (XLT4), and bismuth sulfite agar (BS), also were compared for SE isolation. One hundred twenty-eight environmental drag swab samples were collected from two previously identified SE-positive chicken flocks in two U.S. states and analyzed in parallel using the Reveal test and the FDA culture method. Twenty-five samples (19.5%) yielded SE when the Reveal test was used, and 23 samples (18.0%) were positive for SE by the FDA culture method. No significant difference in efficacy (P = 0.527) was found between the two methods. The Reveal test had a sensitivity, specificity, and accuracy of 83, 94, and 92%, respectively. Overall, a significantly greater number of positive samples was obtained after enrichment in RV compared with TT. XLT4 and BGN were more efficient than BS for isolating SE. However, no single method or medium successfully recovered SE from all SE-positive environmental samples.  相似文献   

5.
目的结合不同标准和参数,对沙门氏菌商品化检测试剂盒进行评价。方法用52个血清型的64株沙门氏菌和20个种属的30株非沙门氏菌,对沙门氏菌商品化核酸检测系统进行包容性和排他性评价;通过与我国国标方法的平行检测,使用20个食品样本对沙门氏菌商品化核酸检测系统进行方法一致性评价。结果商品化检测系统对沙门氏菌的平均检出限为1.51×103 CFU/m L,灵敏度为100%;对30株非沙门氏菌进行检测,商品化核酸检测系统检测结果均为阴性,特异性为100%;对生肉和熟肉制品食品样品进行沙门氏菌检测,沙门氏菌商品化核酸检测系统与国标方法的一致性均在95%以上,均无显著性差异(P0.05),准确度为100%。结论沙门氏菌商品化检测系统以其快速、简便、准确、不需要大型仪器,值得在食品微生物检测行业推广。  相似文献   

6.
There is a need for standardised, robust, reproducible molecular and culture methods to achieve clarification of the inactivation of Mycobacterium avium subsp. paratuberculosis (Map), the causative microbial agent of Johne's disease, in (faecally) contaminated milk and other food products such as meat. This study assessed the performance of a commercially available Map DNA extraction kit for milk Adiapure and accompanying PCR detection kit Adiavet alongside 'in-house' molecular and culture methods in an inter-laboratory ring trial using raw milk spiked with Map-infected faeces. The combined Adiapure-Adiavet Map DNA extraction and detection kit consistently detected 30 copies of IS900 (equivalent to approximately 2 cells) ml(-1) raw milk, when used in four different laboratories. Improvements in sensitivity and ease of use for 'in-house' Map detection were observed when the Adiapure extraction kit was combined with 'in-house' detection assays. Detection by real-time PCR methods, using the commercial extraction and detection systems, resulted in an overall detection rate of 100%, 90%, 85% and 25% for respective Map concentrations of 300, 30, 3 and 0.3 copies of IS900ml(-1) raw milk. Map, at 300 copies of IS900 (equivalent to approximately 20 Map cells) ml(-1) raw milk, was recovered from all samples cultured in mycobacteria growth indicator tube (MGIT) medium, from 10 of 12 samples on Herrold's egg yolk medium (HEYM) and not recovered from any samples using BACTEC medium. In conclusion, the Adiapure DNA extraction kit allows for sensitive and easy detection of Map in raw milk. The extraction method can form a candidate part of essential methodology and real-time PCR can further increase the sensitivity of the detection method. Moreover, MGIT medium is promising for culture-dependent detection of Map from raw milk.  相似文献   

7.
建立肠炎沙门氏菌(Salmonella enteritidis)的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)方法,实现对肠炎沙门氏菌的快速检测.通过针对肠炎沙门氏菌血清型特异性基因lygD设计LAMP内外引物对,优化LAMP扩增反应条件,采用包括肠炎沙门氏菌在内的10种不同菌株进行LAMP引物特异性检测;通过系列梯度稀释肠炎沙门氏菌菌液进行LAMP扩增,计算检出限;并对鲜鸡蛋模拟样本进行LAMP检测.结果表明LAMP法可快速特异地检测出肠炎沙门氏菌;细菌培养液检出限为2.33×101 cfu/mL,鲜鸡蛋模拟样品为2.67×l01cfu/mL.该方法反应灵敏度高,可用于食品中肠炎沙门氏菌的快速检测.  相似文献   

8.
We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.  相似文献   

9.
目的 对试剂盒检测苏丹红I 的方法进行改进,提高试剂盒检测方法的灵敏度和准确性。方法 样品由磁性聚吡咯材料预处理后使用苏丹红I试剂盒进行检测。结果 结合磁固相萃取的试剂盒检测方法,提高了对苏丹红I的检测灵敏度,并将其用于食品中苏丹红I的检测,方法检出限比直接用试剂盒检测降低了3倍。结论 由于磁固相萃取样品前处理方法简单、快速、溶剂用量少,将其与试剂盒检测联用,适于食品基质中苏丹红I的现场检测,能有效避免试剂盒筛查方法的漏检情况,减少假阴性结果的出现。  相似文献   

10.
目的验证沙门氏菌、非沙门菌及阳性核酸模板对MICROFAST?沙门氏菌核酸检测试剂盒(PCR-探针法)的特异性和稳定性,同时比对试剂盒法与GB 4789.4—2016培养法定性检测结果的一致性。方法用实验室保存的30株沙门氏菌和15株非沙门氏菌菌株验证MICROFAST?沙门氏菌核酸检测试剂盒(PCR-探针法)的特异性。通过人工添加不同浓度沙门氏菌对30个乳制品样本,采用国家标准法和试剂盒法同时检验,探究方法的一致性。选择制备好的5份阳性核酸模板,每个模板分别使用3个批次的试剂盒进行检测,对实验结果进行重复性和显著性分析,确定不同试剂盒批次间是否存在显著性差异。结果 30株沙门氏菌菌株和15株非沙门氏菌菌株的特异性检测结果表明,MICROFAST?沙门氏菌核酸检测试剂盒(PCR-探针法)对沙门氏菌的特异性符合预期。人工添加的阳性样本检测结果表明,在乳制品样本范围内,试剂盒的假阳性率与假阴性率为0。3个批次的试剂盒对5份阳性模板检测结果之间没有显著性差异,变异系数CV均小于1%。结论该试剂盒方法与国家标准法相比,具有操作简单、快速等优势,适合乳制品加工过程微生物快速检测。  相似文献   

11.
For Salmonella Enteritidis (SE) detection, shell eggs have been homogenized with stomachers, with electric blenders, and by hand massaging. However, to date, there have been no published reports addressing whether the method of homogenization affects the recovery of SE from raw eggs. Three inoculum levels (10, 126, and 256 SE cells per pool of 10 eggs) were used to conduct three experiments. The 10-egg pools were homogenized by one of four homogenization methods--mechanical stomaching, electric blending, hand massaging, and hand stirring-for 30 s. The homogenized eggs were then incubated at 37 degrees C, and SE colonies were enumerated after 24 and 48 h of incubation. After 24 h of incubation, no SE was recovered from egg samples from stomached or electrically blended pools inoculated with <10 cells, while levels of 106 CFU/ml were found for samples from whipped or hand-massaged pools inoculated with <10 cells. Similarly, after 24 h of incubation, the numbers of SE cells recovered from hand-massaged or hand-stirred egg pools inoculated with 126 cells were significantly larger than the numbers recovered from stomached or electrically blended egg pools inoculated with 126 cells. The number of SE cells recovered from samples homogenized with a blender was still significantly smaller than the numbers recovered from samples homogenized by the other three methods when the inoculum level was increased to 256 CFU per pool. However, the SE count for all samples approached 9 log10 CFU/ml after 48 h of incubation. It is concluded that the detection of small SE populations in shell egg samples could be improved with the use hand massaging and hand stirring for homogenization.  相似文献   

12.
通过对标准菌株和人工污染沙门氏菌的食品样品进行检测,评价IQ-Check Salmonella Ⅱ试剂盒方法。该研究采用试剂盒方法对50株不同血清型的沙门氏菌和50株非沙门氏菌进行检测,分析该方法的灵敏性和特异性;通过对人工污染沙门氏菌食品样品(包括液体奶、婴幼儿配方乳粉、肉及肉类制品)的检测,评价试剂盒方法与GB 4789.4-2016方法的一致性。实验结果表明:当菌浓度在103 CFU/mL及以上,试剂盒方法对50株沙门氏菌实现全部检出,其对不同血清型沙门氏菌检出限的平均值为6.98×102 CFU/mL。试剂盒方法对50株非沙门检测结果均为阴性,说明试剂盒特异性较好。试剂盒方法与GB方法对人工污染沙门氏菌的食品样品阳性检出率分别为98.77%(161/163)和96.32%(157/163);相对准确度为:96%、99%、97%,总体准确度为97.33%;相对灵敏度为:96.22%、100%、100%,总体灵敏度为98.73%;相对特异性为:95.74%、98.15%、92.86%,总体特异性为95.80%。参照ISO 16140进行方法一致性分析,结果表明两方法在统计学上无显著性差异。该研究表明该试剂盒方法具有高灵敏性和特异性强的特点,在人工染菌食品样品检测中与GB方法呈高度一致性,值得在食品沙门氏菌快速检测中推广应用。  相似文献   

13.
Immunochemical-based methods for the detection of Salmonella in food can be complicated by the presence of closely related, immunocrossreactive non-Salmonella species in the sample that may cause false-positive results. To circumvent this problem, specific bacteriophages against immunocrossreactive, non-Salmonella bacteria were used in the sample enrichment step to suppress their growth and improve the performance of an immunochromatographic strip-based detection method for Salmonella. Cross-reactive bacteria were isolated from various food sources and were characterized with a panel of Salmonella somatic O antigen-specific monoclonal antibodies. These cross-reactive bacteria were primarily Citrobacter spp. and Escherichia coli with serology shared with Salmonella serogroups B, D, and F. These bacteria were used as hosts for the isolation of specific lytic bacteriophages. When formulated with the primary enrichment, the bacteriophage cocktail significantly reduced false positives with a broadly reactive immunochromatographic test strip. This was demonstrated in both artificially and naturally contaminated meat. False positives in naturally contaminated beef samples were reduced from 32 of 115 samples tested to zero. In raw meat and poultry with a relatively high bioburden (>10(5) CFU/g), the use of the bacteriophage-based enrichment procedure gave improved recovery of Salmonella compared with the conventional culture-based reference method. This was observed when coupled to either test strip-based or selective agar-based detection. The use of specific bacteriophages for the control of immunocrossreactive and competitive microflora during the food sample enrichment step provides a new approach for enhancing the performance of both immunological- and cultural-based detection methods.  相似文献   

14.
Rapid and molecular technologies such as enzyme-linked immunosorbent assay (ELISA), PCR, and lateral flow immunoprecipitation can reduce the time and labor involved in screening food products for the presence of pathogens. These technologies were compared with conventional culture methodology for the detection of Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7 inoculated in raw and processed meat and poultry products. Recommended protocols were modified so that the same enrichment broths used in the culture methods were also used in the ELISA, PCR, and lateral flow immunoprecipitation assays. The percent agreement between the rapid technologies and culture methods ranged from 80 to 100% depending on the pathogen detected and the method used. ELISA, PCR, and lateral flow immunoprecipitation all performed well, with no statistical difference, compared with the culture method for the detection of E. coli O157:H7. ELISA performed better for the detection of Salmonella, with sensitivity and specificity rates of 100%. PCR performed better for the detection of Campylobacter jejuni, with 100% agreement to the culture method. PCR was highly sensitive for the detection of all the foodborne pathogens tested except Listeria monocytogenes. Although the lateral flow immunoprecipitation tests were statistically different from the culture methods for Salmonella and Listeria because of false-positive results, the tests did not produce any false negatives, indicating that this method would be suitable for screening meat and poultry products for these pathogens.  相似文献   

15.
A high-throughput, rapid method was devised for the detection of Salmonella Enteritidis in egg products. For each target organism, preenrichment in nutrient broth was followed by selective enrichment in Rappaport-Vassiliadis soya peptone and tetrathionate brilliant green broths or by plating on modified semisolid Rappaport Vassiliadis (MSRV) agar medium. The presence of Salmonella Enteritidis was determined by subjecting portions of the selective broth cultures or swarming growth on MSRV medium to an enzyme-linked immunosorbent assay (ELISA) procedure using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for lipopolysaccharide (LPS) antigens. Sample extracts were reacted with polymyxin-coated microwells, and captured LPS antigens were detected immunoenzymatically with a commercially available Salmonella factor O9-specific antibody. The polymyxin-ELISA was 100% sensitive and 100% specific for Salmonella strains bearing the O9 antigen. When the ELISA was combined with enrichment using either the selective broths or plating on MSRV medium, the system was an effective means for detection of Salmonella Enteritidis in artificially inoculated egg products. The polymyxin-ELISA is a simple and inexpensive assay for group D salmonellae (including Salmonella Enteritidis) in a convenient 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.  相似文献   

16.
沙门氏菌核酸检测试剂盒的评价   总被引:1,自引:0,他引:1  
采用室内技术参数验证和协同实验的方式对商品化沙门氏菌核酸检测试剂盒在食品中沙门氏菌核酸检测上的包容性、排他性、灵敏度、特异性、准确度、检测限、耐变性、批间变异进行评价。评价结果表明,试剂盒检测结果与国家标准方法检测结果总体上无显著差异,能满足食品样品的检测要求;但对蔬菜制品的灵敏度较其他食品低,与国家标准方法有显著差异,因此该商品化试剂盒不适用于蔬菜制品中沙门氏菌核酸的检测。  相似文献   

17.
The 1-2 Test is a rapid culture test for the detection of motile Salmonella. The aim of this study was to evaluate the 1-2 Test for its ability to detect Salmonella in swine feces following preenrichment and selective enrichment. Pooled pen fecal samples (n = 118) and pig rectal swabs (n = 51) were cultured for Salmonella by the 1-2 Test, which was compared with the standard isolation protocol currently used in our laboratory. In addition, pen fecal samples known to be free of Salmonella were spiked with various concentrations of Salmonella enterica subsp. enterica serovar Typhimurium and cultured by both methods to determine the minimum number of organisms needed to produce a positive result. When naturally contaminated pen feces and rectal swabs were used, results obtained with the standard culture method were similar to those obtained with the 1-2 Test. However, the 1-2 Test did outperform the standard culture method when the spiked feces samples were tested (chi2 = 4.00). The test kit reduced the time and materials required for the detection of Salmonella in swine feces. The results of this study indicate that the 1-2 Test is an accurate method for monitoring Salmonella in swine feces.  相似文献   

18.
目的对市场上6种商品化比色法测食品中铝残留量快速检测试剂盒(以下简称铝快检试剂盒)进行质量评价;为食品安全监管中铝快检试剂盒的选择和使用提供参考依据。方法分别用铝快检试剂盒和GB5009.182-2017《食品安全国家标准食品中铝的测定》分光光度法对样品中的铝残留量进行测定,验证试剂盒检测的准确性,分析铝快检试剂盒的假阳性率、假阴性率、检出限、符合率,对其进行质量评价。结果试剂盒A、B、D符合率在95%以上,且假阴性率小于10%;试剂盒C、F假阴性率达50%左右;5种试剂盒的检出限与试剂盒标识不相符;试剂盒C、E检测结果不易判定;试剂盒A、B、F试剂装量不足,试剂盒C、F外包装与说明书贮藏条件不一致。结论快速检测试剂盒在使用前,有必要通过假阳性率、假阴性率、检出限、符合率等指标对试剂盒检测结果的可靠性验证。  相似文献   

19.
Mixed raw egg contents were inoculated with approximately 10 CFU of Salmonella Enteritidis and supplemented with 0 to 7 mg of FeSO4 per g of egg contents. Egg contents were then incubated at 37 degrees C, and Salmonella Enteritidis colonies were enumerated for up to 106 h. Iron supplementation significantly enhanced the growth of Salmonella Enteritidis. Within the first 24 h of incubation, the optimum iron level for Salmonella Enteritidis growth in egg contents was between 0.2 and 2 mg of FeSO4 per g of egg contents. After 24 h of incubation at 37 degrees C. Salmonella Enteritidis counts in eggs supplemented with 0.5 mg of FeSO4 per g of egg contents consistently reached approximately 1 x 10(9) CFU/ml, whereas Salmonella Enteritidis counts in eggs without iron supplementation varied from less than 5 CFU/ml to 8.4 x 10(6) CFU/ml. A 3 by 3 factorial design was used to study the effect of type of preenrichment and level of iron supplementation on the growth of Salmonella Enteritidis in egg contents. No significant differences in Salmonella Enteritidis counts between preenrichment and nonpreenrichment treatments were observed when egg contents were supplemented with 0.5 mg of FeSO4 per g of egg contents. It was concluded that preenrichment was not necessary for isolation of Salmonella Enteritidis from eggs. The effect of iron supplementation on the sensitivity of detection by the direct plating method was investigated. The direct plating method detected a significantly higher percentage of Salmonella Enteritidis in raw egg contents supplemented with 0.5 mg of FeSO4 per g of egg contents (90%) than in raw egg contents without iron supplementation (63.3%).  相似文献   

20.
目的采用乳铁蛋白快速检测试剂盒,通过酶联免疫吸附法定量测定婴儿配方奶粉中的乳铁蛋白含量,并验证该方法的准确性、稳定性和可靠性。方法按照试剂盒操作说明制作标准曲线,选取乳铁蛋白阴性样品进行添加回收实验,验证方法的准确性;选取一个乳铁蛋白阳性样品,重复检测6次,计算结果偏差,验证方法的稳定性;选取12个乳铁蛋白阳性样品,分别采用试剂盒方法和高效液相色谱法进行检测,对比2种方法的检测结果,验证试剂盒方法的可靠性。结果试剂盒检出限为2 mg/100 g;试剂盒检测方法回收率在101.6%~109%;相对标准偏差(relative standard deviation,RSD)为5.34%;试剂盒方法检测结果与标签值的偏差为-6.90%~6.45%,和高效液相色谱法检测结果的偏差为-6.45%~6.9%,说明试剂盒方法具有很好的可靠性。结论乳铁蛋白试剂盒检测方法灵敏度高、操作简单,能快速准确地测定婴儿配方奶粉中的乳铁蛋白含量。  相似文献   

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