环介导等温扩增快速检测B群链球菌的临床应用

目的建立采用环介导等温扩增（LAMP）技术从临床标本中快速检测B群链球菌的方法。方法根据美国国家生物信息中心上提交的B群链球菌的cfb基因序列（登陆号X72754）设计特异LAMP引物,以热裂解法提取标本中细菌的DNA,然后以LAMP技术扩增cfb基因来鉴定其是否为B群链球菌。在采用LAMP技术检测B群链球菌的同时,以选择性培养法和PCR技术平行检测相同标本,并将3种方法的检测结果比较。结果在176例阴道拭子和176例直肠拭子中,选择性培养法检测到的B群链球菌例数为49和61、LAMP法检测到的B群链球菌例数为49和59,PCR法检测到的B群链球菌例数为48和58。以选择性培养法为金标准,LAMP法检测B群链球菌的灵敏度和特异度分别为96．7％和100％,PCR法检测B群链球菌的灵敏度和特异度分别为95．1％和100％。LAMP法检测B群链球菌的时间为2h左右,模拟菌株的检测结果显示该方法的最低检测限为10^2CFU／mL。结论该方法灵敏度高,特异性强,操作方便简单,适合从临床标本中快速检测B群链球菌。     

丙型肝炎病毒分片段抗体检测蛋白质芯片的制备及临床评价

为了制备丙型肝炎病毒分片段抗体检测蛋白质芯片,并对其临床应用价值进行评价,将基因工程表达的丙型肝炎病毒分片段抗原,点至经特殊处理的玻片上,制成蛋白质芯片.收集来自三家临床单位用于临床验证的905份血清标本.分别用丙肝病毒分片段抗体检测蛋白质芯片、ELISA丙肝病毒抗体检测试剂进行检测.部分样本同时采用进口RIBA抗体检测试剂进行了检测,分别比较蛋白质芯片法与ELISA法以及RIBA试剂的符合率.结果表明：a.905份血清标本,ELISA法检出阳性294份,阴性611份.阳性标本用蛋白质芯片法检测,融合抗原292份显示阳性结果、2份阴性结果,根据蛋白质芯片的核心抗原,以及NS3, NS4,NS5分片段抗原综合判断确定阳性样本288份阳性,阴性样本2份,4份样本结果不确定.ELISA法检出的611份阴性标本用两种蛋白质芯片法检测,检出阴性均为611份.两种蛋白质芯片法与ELISA法的阳性符合率分别为99.3%和98.9%,与ELISA法的阴性符合率均为100%.用RIBA 试剂检测6份ELISA法为阳性,蛋白质芯片法为非阳性的样本,结果均为非阳性.b.290份经 RIBA试剂确认的阳性标本104份,单片段阳性标本66份,阴性标本120份,用蛋白质芯片法检测,检出阳性标本103份,单片段阳性标本61份,阴性标本126份,二者具有很高的符合率（P＞0.01）.丙型肝炎病毒分片段抗体检测蛋白质芯片,检测灵敏度和特异性高于ELISA法,对血清样本的确认程度与进口的RIBA试剂高度一致,具有操作简便,费用低廉的特点,是一种新型、高效的体外诊断试剂.     

Evaluation of different storage methods to characterize the fecal bacterial communities of captive western lowland gorillas (Gorilla gorilla gorilla)

Freezing is considered to be the best method for long-term storage of bacterial DNA from feces; however this method cannot be usually applied for samples of wild primates collected in the challenging conditions of the tropical forest. In order to find an alternative conservation method of fecal samples from wild great apes, we compared freezing with other fixation methods. Fecal samples from 11 captive gorillas (Gorilla gorilla gorilla) from three Czech Zoos were stored using freezing, RNA Stabilization Reagent (RNAlater), and 96% ethanol. Subsequently, the samples were examined using culture-independent methods (PCR-DGGE, and Real-time PCR) to qualitatively and quantitatively assess fecal microbiota composition and to compare differences among the storage methods. Noticeably, freezing samples resulted in the highest recoveries of DNA. No significant differences in DNA recovery were found between freezing and using RNAlater; however, significantly lower DNA concentrations were recovered from samples stored in 96% ethanol. Using PCR-DGGE we found that either 96% ethanol, RNAlater or freezing were suitable for preserving bacterial DNA; however fingerprints obtained from RNAlater storage were more similar to those obtained from the frozen method; in comparison to the patterns resulting from storing samples in ethanol. Using qPCR, frozen samples yielded the highest values of bacterial counts, with the exception of Enterobacteriaceae, which showed the highest numbers using samples stored in ethanol. Sequences of amplicons obtained from PCR-DGGE belonged to the families Clostridiaceae, Lactobacillaceae, Staphylococcaceae, and Lachnospiraceae, phylum Firmicutes; however most amplicons showed sequence similarity to previously uncultured microorganisms. Bacteria belonging to the phylum Firmicutes were the most frequently identified species in the fecal bacterial communities of captive western gorillas. The study showed that RNAlater is an optimal storage method when freezing is not possible.     

A simple modification of the Baermann method for diagnosis of strongyloidiasis

The diagnosis of Strongyloides stercoralis infections is routinely made by microscopic observation of larvae in stool samples, a low sensitivity method, or by other, most effective methods, such as the Baermann or agar culture plate methods. We propose in this paper a practical modification of Baermann method. One hundred and six stool samples from alcoholic patients were analyzed using the direct smear test, agar culture plate method, the standard Baermann method, and its proposed modification. For this modification the funnel used in the original version of the method is substituted by a test tube with a rubber stopper, perforated to allow insertion of a pipette tip. The tube with a fecal suspension is inverted over another tube containing 6 ml of saline solution and incubated at 37 degrees C for at least 2 h. The saline solution from the second tube is centrifuged and the pellet is observed microscopically. Larva of S. stercoralis were detected in six samples (5.7%) by the two versions of the Baermann method. Five samples were positive using the agar culture plate method, and only in two samples the larva were observed using direct microscopic observation of fecal smears. Cysts of Endolimax nana and Entamoeba histolytica/dyspar were also detected in the modification of Baermann method. Data obtained by the modified Baermann method suggest that this methodology may helps concentrate larvae of S. stercoralis as efficiently as the original method.     

Degradation of bacterial cyclopropane acids with boron trihalide reagents

The cellular fatty acid compositions of Pseudomonas diminuta UC 501 and Streptococcus mutans OMZ-61 were compared in samples processed by a saponification-methylation procedure (method S) and in samples processed by a transesterification procedure (method T). All samples were heated in an inert atmosphere of nitrogen. The major acids found in samples of P. diminuta treated by method S included 16:0 (palmitic), 18:1 (octadecenoic) and 19 cyc (19 carbon cyclopropane acid). Those found in samples of S. mutans treated by the same method included 16:0, 18:1. 18:0 (stearic), 20:1 (eicosenoic), 20:0 (eicosanoic), 19 cyc and 21 cyc. When method T was used to process samples of both cultures, the cyc acids were degraded and artifacts were produced. Transesterification with boron trihalide reagents is therefore not recommended for routine analysis of bacterial fatty acids.     

Identification of Legionella spp. in Environmental Water Samples by ScanVIT-Legionella™ Method in Spain

Rapid and more sensitive methods for the detection and quantification of viable Legionella cells have been developed. In this paper, a comparative analysis of environmental water samples using the ScanVIT-Legionella? method and the traditional “gold standard” method of culturing is realised indicating the usefulness of the ScanVIT method. The ScanVIT-Legionella? method was performed on environmental water samples from different locations of Huesca region (Spain). Legionella micro-colonies should appear green colour and Legionella pneumophila micro-colonies appear red. Twenty-one environmental water samples analysed by standard culture plus five control samples (Two sterile water samples with Legionella as positive controls and three sterile water samples as negative controls). All of them were used to apply ScanVIT-Legionella? method. From of 21 environmental samples eleven were positive, six negative with both methods and four samples were negative for culture method and positive for ScanVIT-Legionella? method. The positive control samples were positive and the negative were negative for both methods. A comparative analysis of the results obtained with two methods showed a strong positive determination coefficient (R² = 0.99753). The results demonstrate the usefulness of the ScanVIT-Legionella? method as a rapid diagnostic tool in order to provide a diagnosis as quick as possible. ScanVIT-Legionella? method offers a series of advantages such as quickly diagnosis, higher sensitivity and the possibility to identify Legionella spp. and L. pneumophila simultaneously.     

Rapid detection of Salmonella spp. in food by use of the ISO-GRID hydrophobic grid membrane filter.

A rapid hydrophobic grid-membrane filter (HGMF) method was developed and compared with the Health Protection Branch cultural method for the detection of Salmonella spp. in 798 spiked samples and 265 naturally contaminated samples of food. With the HGMF method, Salmonella spp. were isolated from 618 of the spiked samples and 190 of the naturally contaminated samples. The conventional method recovered Salmonella spp. from 622 spiked samples and 204 unspiked samples. The isolation rates from Salmonella-positive samples for the two methods were not significantly different (94.6% overall for the HGMF method and 96.7% for the conventional approach), but the HGMF results were available in only 2 to 3 days after sample receipt compared with 3 to 4 days by the conventional method.     

基于微滴式数字PCR检测肠癌病人游离环状DNA KRAS突变的新方法

基于微滴式数字聚合酶链式反应(Droplet digital polymerase chain reaction,dd PCR)设计一种检测肠癌游离循环DNA(Circulating cell free DNA,cf DNA)中KRAS(V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog)基因突变的新方法并评估其灵敏度和准确性。根据肠癌病人KRAS基因的突变类型设计并合成,采用dd PCR扩增并评估其灵敏度和准确性;根据AMRS-PCR引物设计原理设计KRAS基因的实时定量PCR扩增引物并评估其准确性,进而比较dd PCR和q PCR二者之间的优缺点;最后针对52例肠癌病人的cf DNA采用dd PCR进行检测,研究dd PCR在cf DNA KRAS基因突变检测的应用。成功使用dd PCR和q PCR两种方法对KRAS野生型及7种突变型建立检测方法,使用质粒标准品及实际样品验证该两种方法可行并对其假阳性率、线性范围及检测下限等性能进行了评价,最后成功对52例临床患者和20例正常人的血浆cf DNA样本进行检测,临床灵敏度为97.64%,临床特异性为81.43%。dd PCR的检测性能优于q PCR,LOD达到个位数DNA拷贝,最低可确认突变浓度达到0.01%–0.04%。样本提取效率在方法学建立中也十分重要,直接影响到灵敏度和Cut Off值的判定。临床患者检测结果显示其KRAS突变率接近报道水平。     

Comparison of Clark's presence-absence test and the membrane filter method for coliform detection in potable water samples.

A total of 2,601 water samples from six different water systems were tested for coliform bacteria by Clark's presence-absence (P-A) test and by the membrane filter (MF) method. There was no significant difference in the fraction of samples positive for coliform bacteria for any of the systems tested. It was concluded that the two tests are equivalent for monitoring purposes. However, 152 samples were positive for coliform bacteria by the MF method but negative by the P-A test, and 132 samples were positive by the P-A test but negative by the MF method. Many of these differences for individual samples can be explained by random dispersion of bacteria in subsamples when the coliform density is low. However, 15 samples had MF counts greater than 3 and gave negative P-A results. The only apparent explanation for most of these results is that coliform bacteria were present in the P-A test bottles but did not produce acid and gas. Two other studies have reported more samples positive by Clark's P-A test than by the MF method.     

Pyrosequencing of a short fragment of the amelogenin gene for gender identification

We report a pyrosequencing method for detecting a short amelogenin fragment to aid the gender identification. The PCR products (44/45 bp), including primers and target sequence (4/5 bp) consisting of three point mutations and one indel mutation, were sequenced by the pyrosequencing method. 100 randomly chosen DNA samples of healthy donors were analyzed with this method, and all of them were correctly typed. The sensitivity of the technique was 0.5 ng template DNA. No specific peak was found in any detected animals or organisms except for monkey. For blood samples that were left outside for 26 weeks and DNA degraded artificially by digesting with DNaseI, this method gave more accurate results than the conventional method. Moreover, four bone samples analyzed using the method gave clear pyrograph. This method is easy, quick, cheap and suitable for high-throughput analysis, especially for identifying the gender of highly-degraded DNA samples.     

Comparison of Clark's presence-absence test and the membrane filter method for coliform detection in potable water samples

A total of 2,601 water samples from six different water systems were tested for coliform bacteria by Clark's presence-absence (P-A) test and by the membrane filter (MF) method. There was no significant difference in the fraction of samples positive for coliform bacteria for any of the systems tested. It was concluded that the two tests are equivalent for monitoring purposes. However, 152 samples were positive for coliform bacteria by the MF method but negative by the P-A test, and 132 samples were positive by the P-A test but negative by the MF method. Many of these differences for individual samples can be explained by random dispersion of bacteria in subsamples when the coliform density is low. However, 15 samples had MF counts greater than 3 and gave negative P-A results. The only apparent explanation for most of these results is that coliform bacteria were present in the P-A test bottles but did not produce acid and gas. Two other studies have reported more samples positive by Clark's P-A test than by the MF method.     

Rapid identification and quantification of Collinsella aerofaciens using PCR

The number and incidence of Collinsella aerofaciens in the human intestine are the highest among Gram-positive non-spore-forming bacilli. Identification of this species is very difficult and requires considerable time. A PCR-based identification system using C. aerofaciens-specific primers is described. Using this PCR method, we identified 181 C. aerofaciens-like species isolated from human feces. These 181 strains were identified using the traditional method in past studies. Results of both methods matched. The direct detection method was performed using human feces samples from seven adults. Nested PCR was applied directly to the samples and all seven samples were positive. Quantification studies were performed using LightCycler?trade mark omitted?. The assay uses a double-stranded DNA dye to continuously monitor product formation and in a short time is able to quantify samples to 5 log units in concentration.     

Ubiquinone binding protein used for determination of coenzyme Q

The conventional method of assaying for the ubiquinone (CoQ) content of biological samples is to partition CoQ into an organic phase and separate it from contaminants by high-performance liquid chromatography (HPLC). HPLC is an accurate method of quantifying CoQ content but is not ideal for routine clinical analyses. This paper describes the development of a rapid method for assaying the CoQ content of biological samples based on the binding of CoQ to a CoQ binding peptide. The 14-amino acid binding peptide was chemically synthesized, and conditions for immobilizing the peptide on microfuge tubes were established. CoQ could be selectively bound to the immobilized peptide, eluted, and determined spectrophotometrically. Limits of detection for the method were 0.25 to 5 nmol CoQ. To test biological samples, CoQ was isolated from cultures of Saccharomyces cerevisiae grown in oleic acid medium. The recovery of CoQ samples using the binding assay ranged from 99 to 102% of the values obtained with HPLC. The assay described here provides an inexpensive, rapid method for determining the CoQ content of large numbers of biological samples in a variety of laboratory settings.     

一种利用Profile-1生物发光仪快速测定土壤中微生物量的改良方法

开发了一种利用Profile-1生物发光仪测定土壤中微生物量的改良方法,并以此方法分别测定了标准大肠杆菌茵液以及3种不同类型的土壤(九段沙湿地土壤,崇明东滩大田土壤和崇明实验地改良土壤)的微生物量,并将结果与Profile-1生物发光仪自带的标准分析方法以及传统的菌落计数法进行比较。结果显示,改良的ATP提取方法(BAB改良分析法)和Profile-1生物发光仪自带的标准分析方法都可用于液体样品中微生物量的测定,其灵敏度和准确度无显著差异(P0.05)。但在测定土壤样品时,菌落计数法测定结果大约占BAB改良分析法测定结果的1%～5%,占Profile-1生物发光仪自带的标准分析方法的测定结果的22%～99%。这表明在分析土壤样品时,BAB改良分析法较Profile-1生物发光仪自带的标准分析方法的ATP提取效率更高,可显著提高仪器检测土壤样品的灵敏度和可靠性,因此可有效应用于各类土壤的微生物量的监测,为土壤环境监控提供微生物量的可靠数据。     

Validation of High Resolution Melting Analysis (HRM) of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples: Comparison with Culture and MALDI-TOF Based Identification

AimCandida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method), by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method) to establish the presence of Candida species in three different types of clinical samples.ResultsFor 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample.DiscussionMost of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method compensate for its disadvantages.     

Evaluation of real-time PCR vs automated ELISA and a conventional culture method using a semi-solid medium for detection of Salmonella

AIMS: Evaluation of iQ-Check PCR Salmonella for Salmonella detection in artificially and naturally contaminated food and environmental field samples. METHODS AND RESULTS: Artificially contaminated samples (poultry meat and ground red meat) subjected to cold- and freeze-stress, and 120 naturally contaminated samples (swabs and meat) were tested for Salmonella using the diagnostic semi-solid Salmonella medium (DIASALM) method, the Vidas assay and the iQ-Check PCR assay after 24 h enrichment in buffered peptone water. CONCLUSIONS: Both the iQ-Check PCR and the Vidas assay provide a rapid and user friendly screening method for detection of Salmonella. False negative samples were obtained for the inoculated samples using both the iQ-Check PCR assay and the Vidas method when Salmonella cells were severely stressed. In total 45 of 120 naturally contaminated field samples showed Salmonella positive using the DIASALM method. The agreement percentage with the DIASALM method was respectively 92% for the iQ-Check PCR and 95% for the Vidas method. SIGNIFICANCE AND IMPACT OF THE STUDY: False-negative samples were obtained for the inoculated samples using both the iQ-Check PCR assay and the Vidas method when Salmonella cells were severely stressed, e.g. freezing at -18 degrees C for 7 days. Of the 120 naturally contaminated field samples 45 showed Salmonella positive using the DIASALM method. The agreement percentage with the DIASALM method was 92% for the iQ-Check PCR and 95% for the Vidas method respectively.     

一种检测kras基因突变的新型TB-ARMS qPCR方法的建立

在荧光定量PCR基础上建立一种简单有效并且高度灵敏的TB-ARMSkras基因突变检测方法,并对其检测性能进行评估,探讨其临床应用价值。针对kras基因8种常见的点突变类型,通过设计并优化突变特异性引物、野生型特异性封闭引物并综合应用突变富集扩增反应条件等多种手段,提高点突变检测的灵敏度和特异性,采用已知野生型基因组样品和构建的突变质粒作为标准品,进行方法学评价;通过对临床样本的检测及与现有商品化试剂盒的比较进行性能验证;通过对术前血浆和配对组织样品的对比检测,评估方法是否适用于血液样本的检测。建立了TB-ARMS kras突变检测的新方法,能检测的最低突变率可达到0.01%。通过综合采用野生型特异性封闭引物和突变富集扩增条件等方法证明了其0.01%的突变检测灵敏度。检测准确性优于现有商品化试剂盒,血浆DNA TB-ARMS qPCR检测结果与配对组织DNA测序结果相符合。因此,TB-ARMS kras基因突变检测方法具有广泛的临床应用价值,既适用于临床组织样品的检测,也可应用于液体活检。     

环介导等温扩增技术在食品中痢疾志贺氏菌快速检测

【目的】探讨、优化基于环介导等温扩增技术(LAMP)快速检测常规食品中感染性痢疾志贺氏菌的方法。【方法】在NCBI数据库中搜索获取志贺氏菌的特异性基因高度保守区,设计3对LAMP反应引物,建立、优化该LAMP可视化检测方法,并评价其特异性、灵敏度,同时与PCR检测方法和传统检测方法对比,进行结果统计分析。【结果】5株志贺氏菌标准菌株样品均检测为阳性,11株非志贺氏菌标准菌株样品均检测为阴性,无交叉反应。最低检验限为1.6×101 CFU/反应(或1.6×101 CFU/m L),且经比较,LAMP检测灵敏度比PCR检测高出1个数量级。通过对161份实际样品和人工污染样品进行检测,LAMP检测与传统方法检测结果具有较高的一致性。【结论】LAMP具有检测过程快速简便、检测结果稳定、可靠的特点,适用于对常规食品中感染性痢疾志贺氏菌的高效、快速检测需求。     

Detecting the presence of infectious hepatitis A virus in molluscs positive to RT-nested-PCR

AIMS: The objective of this study was to determine the presence of infectious hepatitis A virus (HAV) in molluscs naturally contaminated with viral HAV-RNA. METHODS AND RESULTS: One hundred and forty-two mollusc samples were analysed for the presence of viral HAV-RNA using RT-nested-PCR; positive samples were then analysed with an integrated method, cell-culture RT-PCR, to detect infectious virus. Viral HAV-RNA was detected in 34.5% of the samples while 12.7% of the total samples were positive for the presence of infectious virus. CONCLUSIONS: The results demonstrate the validity of the screening method (RT-nested-PCR) and the necessity of applying a method that is capable of detecting the presence of infectious HAV. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that in any case, to determine the safety for human consumption, the results of RT-nested-PCR must be confirmed with an integrated cell-culture PCR method.