西替利嗪联合卡介菌多糖核酸对儿童过敏性紫癜的临床疗效分析

目的:观察分析西替利嗪联合卡介菌多糖核酸对儿童过敏性紫癜的临床疗效.方法:将我院从2010年1月至2011年10月我院收治的144例儿童过敏性紫癜患者随机平均分为对照组72例,应用以西替利嗪为主的常规治疗,实验组72例在对照组基础上加用卡介菌多糖核酸,治疗持续3周.检测对比治疗前后患者抗体水平,并统计计算治疗有效率;随访6个月,统计复发情况.结果:两组治疗前后血清T细胞和抗体、补体水平变化结果显示,治疗前组间差异无统计学意义(P＞0.05);治疗后,实验组CD3、CD4和CD16+56上升程度大于对照组,CD8、CD19、IgA和C3、C4下降程度大于对照组,组间差异有统计学意义(P＜0.05);对照组复发率为11.8％(8/68),治疗满意度为85.3％;实验组复发率为5.7％(4/70),治疗满意度为92.9％,组间差异有统计学意义(P＜0.05);对照组痊愈32例,显效25例,好转7例,无效4例,治疗有效率为83.8％;实验组痊愈39例,显效27例,好转3例,无效1例,治疗有效率94.3％,组间差异有统计学意义(P＜0.05).结论:西替利嗪联合卡介菌多糖核酸能有效治疗儿童过敏性紫癜,安全高效,值得推广应用.     

快速鉴定诺卡菌的多重聚合酶链反应的建立

为快速准确鉴定诺卡菌,首先设计针对诺卡菌rpoB、secA1、16S rRNA基因的引物,利用单重聚合酶链反应(PCR)和测序验证引物的特异度,建立多重PCR鉴定系统,在同一反应体系和条件下对44株诺卡菌标准株、44株临床分离株和7株对照株进行扩增。结果显示,利用单对引物对其中2株诺卡菌(标准株DSM43003、临床株CDC 51)进行扩增,出现的条带均为与目的片段长度一致的单一条带,经测序并经基本局部比对搜索工具(BLAST)验证扩增片段为目的基因。建立的多重PCR结果显示,44株诺卡菌标准株中有43株(97.7%)、44株临床分离株中有42株(95.5%)rpoB、secA1、16S rRNA这3条片段均显示,7株对照株均未显示条带。结果提示,本研究建立的多重PCR简单、快速、灵敏度高、特异度好,适用于诺卡菌的快速鉴定。     

鼠伤寒沙门氏菌多重PCR检测方法的研究

分别根据沙门氏菌16S rRNA、质粒毒力基因spvC、致病基因invB、fimA序列设计4对引物,对沙门氏菌株及非沙门氏株菌基因组DNA进行多重PCR检测。结果该方法能检测出6.3×10² 个cfu/ml纯培养的沙门氏菌,人工染菌食品模拟检测结果显示,熟鸡肉初始含菌量为17cfu/g、全脂奶粉为11cfu/g、生牛肉为13.6cfu/g,经过8h增菌,PCR检测为阳性。该体系能鉴定产生多种毒力因子的沙门氏菌,特异性强、敏感性高,为检测和鉴定沙门氏菌株提供了一个新方法。     

多重PCR对真菌性角膜炎主要致病菌的菌属鉴定

目的:建立多重PCR体系对真菌性角膜炎主要致病真菌进行快速诊断并同时进行菌属鉴定的方法。方法:建立两个多重PCR体系(体系1和体系2),对真菌性角膜炎九种主要致病真菌DNA进行检测,观察该体系对真菌临床菌株、人类基因组及其他眼部常见致病微生物DNA的检测结果。结果:体系1对镰孢菌属扩增均产生约360bp的特异产物,对曲霉菌属、牵连青霉菌和新月弯孢菌扩增均产生约470bp的特异产物。体系2对镰孢菌属、曲霉菌属均无特异产物,而对牵连青霉菌产生了360bp的特异产物,对新月弯孢霉产生了300bp的特异产物。根据DNA模板在两个多重PCR体系中扩增出的不同特异条带可将九种真菌分为四个菌属。57株真菌临床菌株中55株的鉴定结果与常规鉴定结果一致。两体系对人类基因组及其他眼部常见致病微生物DNA的扩增结果均为阴性。结论:通过两个多重PCR体系检测可将真菌性角膜炎在菌属水平进行诊断及鉴定。该方法具有快速、简便、特异、灵敏的特点,具有较好的临床应用前景。     

卡介菌多糖核酸对慢性阻塞性肺疾病患者炎症因子水平及免疫功能的影响

目的:分析卡介菌多糖核酸对慢性阻塞性肺疾病患者炎症因子水平及免疫功能的影响。方法:随机将110例慢性阻塞性肺疾病患者分为对照组与观察组,每组55例。对照组采用常规治疗,观察组在常规治疗基础上加用卡介菌多糖核酸治疗,比较两组临床疗效,血清单核细胞样受体4(TLR4)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)、脂质过氧化物(LPO)、超氧化物歧化酶(SOD)、金属蛋白抑制1(TIMP-1)及金属蛋白酶-9(MMP-9)水平,CD4~+、CD4~+/CD8~+、CD8~+水平,第1秒用力呼气容积(FEV1)、肺活量(FVC)。结果:观察组总有效率为96.86%,显著高于对照组81.82%,差异有统计学意义(P0.05)。治疗后,观察组血清TLR4、IL-8、TNF-α、LPO、TIMP-1、MMP-9、HMGB1水平、FEV1、FVC及CD8~+低于对照组,SOD水平、CD4+、CD4+/CD8~+高于对照组,差异均有统计学意义(P0.05)。结论:卡介菌多糖核治疗慢性阻塞性肺疾病的临床疗效肯定,可选择性减轻炎症反应并改善免疫功能。     

红色毛癣菌菌株的特异性PCR鉴定方法研究

目的建立一种快速的红色毛癣菌分子生物学鉴定方法。方法根据红色毛癣菌保守区域-真菌核糖体DNA（rDNA）的转录间隔区（ITS）设计特异性引物,采用上游：ITS19865＇GAC ACC AAG AAA AAA TTC TCT GAA GA3＇,下游：ITS24415＇GTC CTG AGG GCG CTG AA3＇为引物对45株红色毛癣菌、5株须癣毛癣菌和1株紫色毛癣菌菌株的DNA进行PCR扩增,观察产物电泳带型的差异。结果 45株红色毛癣菌均能扩增出目的片段,5株须癣毛癣菌和1株紫色毛癣菌均无目的片段扩增出。结论红色毛癣菌可用特异引物PCR方法快速鉴定。     

RT—nested PCR检测肾综合征出血热患者血清病毒核酸

采用异硫氰酸胍－酚－氯仿（ＡＧＰＣ）一步法提取病毒ＲＮＡ,并依据肾综合征出血热病毒（ＨＦＲＳＶ）核蛋白（ＮＰ）编码基因保守区核苷酸序列合成两对巢式引物,建立了逆转录巢式聚合酶链反应（ＲＴ－ｎｅｓｔｅｄＰＣＲ）检测ＨＦＲＳＶＲＮＡ方法,应用此法对ＨＦＲＳＶ感染的ＶｅｒｏＥ６细胞培养液及ＨＦＲＳ患者血清中的病毒ＲＮＡ进行检测。结果显示,感染细胞培养液及３５例ＨＦＲＳ患者血清均为阳性,正常的ＶｅｒｏＥ６     

用PCR方法扩增深红红螺菌的draT基因

聚合酶链反应⁽¹⁾是最近几年分子生物学领域中一项重大的技术突破。典型的PCR引物内应含50％G+c,且没有自身互补序列,特别是在其3’－末端不应有内部二级结构。引物与靶DNA的退火温度一般为50℃,但当已有的引物不太符合要求时,就要调整退火温度。本文报道了用PCR方法扩增深红红螺菌draT基因,介绍了当引物Gc含量较低时,可采用适当降低退火温度,然后梯度升温的方法获得PCR产物。     

PCR结合酶切-序列比对法鉴定B群脑膜炎奈瑟菌菌株

目的用PCR结合酶切-序列比对法对B群脑膜炎奈瑟菌菌株进行鉴定。方法用玻片凝集法对不同来源的15株B群脑膜炎奈瑟菌菌株进行初步检定,再用PCR结合酶切-序列比对法对上述15株菌株进行进一步鉴定,即用PCR结合酶切法扩增菌株的唾液酸转移酶sia D基因并对PCR产物进行酶切后,用BLAST软件将PCR产物测序结果与Gene Bank中原始sia D序列比对。结果 15株菌株玻片凝集结果均为阳性;15株菌株的PCR产物片段大小均为460 bp;TaqⅠ酶切后,13株菌株的酶切产物片段大小仍为460 bp,其PCR产物测序比对结果与B群脑膜炎奈瑟菌原始sia D序列同源性均达到99%;其余2株酶切产物片段大小约200 bp,与C群脑膜炎奈瑟菌sia D原始基因序列同源性分别为98%和99%。结论 15株菌株经PCR结合酶切-序列比对法鉴定,13株为B群脑膜炎奈瑟菌菌株,2株为C群脑膜炎奈瑟菌菌株;该方法可准确鉴定B群脑膜炎奈瑟菌菌株。     

PCR鉴定肺炎克雷伯菌的强毒性血清型

目的:了解肺炎克雷伯菌强毒性血清型K1、K2、K54和K57型菌株在我国重庆、北京、深圳三地的分布及流行趋势。方法:采用PCR对310株肺炎克雷伯菌临床分离株进行血清型K1、K2、K54和K57检测。结果:310株菌中,K1、K2、K54和K57血清型分别占14.2%、9.4%、6.5%和4.2%;来自呼吸系统标本分离株中的K1血清型菌株在4种检测的强毒血清型中占首位,为呼吸系统总数的17.4%。结论:310株肺炎克雷伯菌的4种强毒性血清型中,K1血清型菌株所占比例高,较为流行。     

Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.     

Simultaneous detection and identification of Bacillus cereus group bacteria using multiplex PCR

Bacillus cereus group bacteria share a significant degree of genetic similarity. Thus, to differentiate and identify the Bacillus cereus group efficiently, a multiplex PCR method using the gyrB and groEL genes as diagnostic markers is suggested for simultaneous detection. The assay yielded a 400 bp amplicon for the groEL gene from all the B. cereus group bacteria, and a 253 bp amplicon from B. anthracis, 475 bp amplicon from B. cereus, 299 bp amplicon from B. thuringiensis, and 604 bp amplicon from B. mycoides for the gyrB gene. No nonspecific amplicons were observed with the DNA from 29 other pathogenic bacteria. The specificity and sensitivity of the B. cereus group identification using this multiplex PCR assay were evaluated with different kinds of food samples. In conclusion, the proposed multiplex PCR is a reliable, simple, rapid, and efficient method for the simultaneous identification of B. cereus group bacteria from food samples in a single tube.     

Preliminary study on the detection of the SARS-CoV specific target cDNA fragments by multiplex PCR

The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The re~sults indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully.     

Fluorescent detection techniques for real-time multiplex strand specific detection of Bacillus anthracis using rapid PCR

Speed is a key area in our development of PCR assays for Bacillus anthracis. We believe that the strand specific detection of amplicons within 10 min is a realistic goal and that this will be achieved through fluorescent in-tube assays. We have used the Idaho LightCycler to study and develop candidate assays for B. anthracis. New strand specific fluorescent methods have been developed and a number of formats have been studied for speed and sensitivity. Internal controls have been developed as a method of improving our assay confidence. In this communication we will introduce the field of rapid PCR whilst discussing previous work in the areas described above, the development of our own rapid assay and a novel internal control system for B. anthracis. This work used PCR assays and hardware that are either commercially available, or have been previously described in open literature publications.     

致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立

目的实现对致病性大肠埃希菌（E．coli）、沙门菌（Salmonella）的同时检测,建立快速灵敏的双重PCR检测方法。方法以致病性大肠埃希菌和沙门菌毒力岛基因为研究对象,根据GenBank发表的大肠埃希菌和沙门菌毒力岛基因序列,分别设计合成了大肠埃希菌毒力岛irpl、irl）2和fyuA,沙门菌毒力岛mgtC、sseL和sopB等6对引物,以禽致病性大肠埃希菌（CVCC1565）菌株和沙门菌（ATCC9150）菌株的核酸混合物为模板,经引物特异性试验,引物组合,成功建立了快速鉴别检测致病性大肠埃希菌和沙门菌的双重PCR方法。结果特异性试验结果显示,引物irpl、irp2和fyuA仅能扩增出大肠埃希菌（CVCC1565）的特异性片段,大小分别是799、414和948bp;引物mgtC、sseL和sopB仅能扩增出沙门菌（ATCC9150）的特异性片段,大小分别是500、269和1000bp。敏感性试验结果表明大肠埃希菌和沙门菌的最低检测限分别为2．2×101CFU／mL和2．0×101CFU／mL。结论本研究建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于致病性大肠埃希菌和沙门菌的联合检测与鉴别诊断。     

Application of the multiplex PCR and PCR-RFLP method in the identification of the Bacillus anthracis

The aim of this study was to apply the multiplex PCR and PCR-RFLP method for the identification of the B. anthracis strains and to distinguish those bacteria from other members of the Bacillus cereus group. The multiplex PCR method enables to detect the virulence factors, i.e. the toxin and the capsule in B. anthracis strains. To do that, the authors have used 5 primer pairs specific for the fragments of lef, cya, pag genes which are present in the pXO1 plasmid and encode the toxin, the cap gene, which is present in the pXO2 plasmid and encodes the capsule, and the Ba813 chromosomal sequence. Among the four B. anthracis strains examined, three contained two plasmids and the Ba813 chromosomal sequence, while the fourth one contained the pXO1 plasmid only, together and the Ba813 chromosomal sequence. Other bacterial species, belonging to the B. cereus group, were also examined: 6 strains of B. cereus, 4 strains of B. thuringiensis and one strain of B. mycoides. The presence of Ba813 chromosomal sequence has been detected in two B. cereus strains. Neither plasmids nor Ba813 chromosomal sequence have been discovered in other B. cereus, B. thuringiensis and B. mycoides strains. The results of the survey indicate that the Ba813 chromosomal sequence does not occur solely in B. anthracis strains. The PCR-RFLP method with the use of SG-749f and SG-749r primers enabled to demonstrate the presence of DNA sequence (SG-749) in B. anthracis, B. cereus, B. thuringiensis and B. mycoides strains. Restriction analysis with enzyme AluI of the SG-749 sequence, has shown the presence of two DNA fragments at the size of about 90 and 660 bp in all B. anthracis strains. The restriction profile obtained was characteristic for B. anthracis strains and it did not occur in other investigated bacterial species belonging to the B. cereus group. It was not observed even in such B. cereus strains in which the presence of Ba813 sequence was discovered and it enabled to differentiate between B. anthracis strains and other closely related species of the B. cereus group.     

应用特异PCR快速鉴定微生物肥料中4种乳酸菌

【目的】植物乳杆菌(Lactobacillus plantarum)、鼠李糖乳杆菌(L.rhamnosus)、嗜酸乳杆菌(L.acidophilus)和德氏乳杆菌(L.delbrueckii)是微生物肥料生产中常用的乳酸菌,它们表型特征相似,若采用传统方法鉴定则费时费力,为准确、快速地鉴定这些种,建立种特异PCR方法。【方法】利用NCBI中Primer-BLAST(引物设计和特异性检验工具),以GenBank数据库中上述菌种的recA和gyrB为靶基因,设计和筛选种特异性引物从而建立相应特异PCR鉴定方法。【结果】经过乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)、片球菌属(Pediococcus)、芽孢杆菌属(Bacillus)、类芽孢杆菌属(Paenibacillus)、短芽孢杆菌属(Brevibacillus)和假单胞菌属(Pseudomonas)7个属24个种共40株标准菌株的实验验证,4个目标种分别扩增出唯一的目的产物,而其他种均无目的扩增产物。采用建立的4种特异PCR方法对产品中分离的16株乳杆菌进行鉴定,结果与16S rDNA序列分析、Biolog鉴定结果一致。【结论】建立的特异PCR鉴定方法均具有较高的种内通用性和种间特异性,可快速、准确的用于微生物制剂中植物乳杆菌、德氏乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌的检测和鉴定,具有较好的应用前景。     

Calibration-curve-free quantitative PCR: a quantitative method for specific nucleic acid sequences without using calibration curves

We have developed a simple quantitative method for specific nucleic acid sequences without using calibration curves. This method is based on the combined use of competitive polymerase chain reaction (PCR) and fluorescence quenching. We amplified a gene of interest (target) from DNA samples and an internal standard (competitor) with a sequence-specific fluorescent probe using PCR and measured the fluorescence intensities before and after PCR. The fluorescence of the probe is quenched on hybridization with the target by guanine bases, whereas the fluorescence is not quenched on hybridization with the competitor. Therefore, quench rate (i.e., fluorescence intensity after PCR divided by fluorescence intensity before PCR) is always proportional to the ratio of the target to the competitor. Consequently, we can calculate the ratio from quench rate without using a calibration curve and then calculate the initial copy number of the target from the ratio and the initial copy number of the competitor. We successfully quantified the copy number of a recombinant DNA of genetically modified (GM) soybean and estimated the GM soybean contents. This method will be particularly useful for rapid field tests of the specific gene contamination in samples.     

A proposal on metrics for identification using nucleic acid sequences

Abstract The need is stressed for attempts to be made to permit diagnostic nucleic acid sequences to be used in a quantitative manner. Sequence differences or binding values should be converted to a distance measure and from this an ultrametric tree should be constructed. A single quantitative determination can yield considerable information about the likely identity of an unknown microorganism when the distance obtained from the sequence is compared with the tree. The concept is illustrated by hypothetical species and genus subsequences, and it is suitable both for successive use of hierarchical subsequences and for automated identification. It is pointed out that entirely specific subsequences for higher taxa may be difficult to discover. These principles will be useful for the future design of diagnostic sequences, including possible application to DNA-DNA pairing.