灰黄青霉Pa-pex11基因对青霉素产生的影响

摘要：【目的】研究灰黄青霉Pa-pex11对青霉素产生的影响。【方法】通过保守序列设计引物从灰黄青霉中克隆了含有pex11同源基因DNA片段,进而通过热不对称交错PCR(Thermal asymmetric interlaced PCR, Tail PCR )扩增得到灰黄青霉中Pa-pex11基因的全序列。利用根癌农杆菌介导的遗传转化（ATMT）系统,在灰黄青霉基因组上导入额外的Pa-pex11基因。对灰黄青霉野生型菌株和转化子进行了青霉素发酵及生物活性测定。通过荧光定量PCR(quantitative     

珊瑚病原菌株XSBZ03和XSBZ14双重PCR检测方法的建立

【目的】建立珊瑚病原菌株XSBZ03和XSBZ14的双重PCR检测方法。【方法】以XSBZ03和XSBZ14的特异靶序列为对象,开展引物设计和双重PCR检测方法的构建,并确定该双重PCR方法的特异性、敏感性及可靠性。【结果】该检测方法可特异识别菌株XSBZ03和XSBZ14,对XSBZ03和XSBZ14基因组DNA样品的检测极限分别为1.7pg/μL和2.0pg/μL;对XSBZ03和XSBZ14在海水样品中的检测极限分别为6×10~3 CFU/mL和8×10~3 CFU/mL。【结论】该方法具有特异性强、灵敏度高等优点,可对由菌株XSBZ03和XSBZ14引起的珊瑚疾病进行准确快速的诊断,为今后开展珊瑚疾病防控和无特定病原的珊瑚移植提供了可靠手段。     

基于16S rDNA基因的谷斑皮蠹PCR检测技术

【目的】谷斑皮蠹是一种重要的检疫性害虫,在新疆周边多个国家分布,口岸检疫人员多次从进境货物中截获谷斑皮蠹,该虫对新疆的农业生产极具威胁。【方法】以谷斑皮蠹的16S rDNA基因为靶序列,用昆虫通用引物对4种供试皮蠹进行PCR扩增,将扩增产物进行克隆和测序,用生物软件设计检测谷斑皮蠹的特异性引物与探针。【结果】设计的特异性引物(TG-SNP-F/TG-SNP-R)及所建立的常规PCR方法能有效检测出谷斑皮蠹,其扩增产物的片段大小为250 bp,灵敏度为3 ng·μL~(-1)。设计的特异性引物(TG-F/TG-R)和探针(TG-probe),以及所建立的实时荧光PCR方法,对谷斑皮蠹的检测特异性强,灵敏度达0.8 fg·μL~(-1)。【结论】建立的常规PCR方法和实时荧光PCR检测方法能够对谷斑皮蠹进行准确鉴定,为口岸检疫人员检测进境货物中携带的谷斑皮蠹提供技术支持。     

应用TaqMan荧光定量PCR检测土拉弗朗西斯菌

目的：利用Roche LightCycler实时定量PCR系统建立一种快速、灵敏、特异的检测土拉弗朗西斯菌的方法。方法：基于TaqMan荧光探针实时定量PCR技术,选择土拉弗朗西斯菌染色体上的特异序列[醇醛酮还原酶（AKR）和外膜蛋白FopA基因]作为检测靶序列,建立土拉弗朗西斯菌实时定量PCR检测方法;评价该检测方法的特异性和灵敏性;采用克隆菌株污染环境土壤来模拟实际样品,评价该检测方法在快速检测与现场检测等实际应用中的表现。结果：优化筛选基因组中的FT-AKR和FT-fopA片段作为检测靶序列,所建立的土拉弗朗西斯菌实时定量PCR检测方法检测克隆菌株质粒的灵敏度均为10个拷贝/每个反应体系;以其他非土拉弗朗西斯菌为模板未出现非特异扩增;模拟环境土壤样品检测灵敏度2个引物对分别为440和960CFU/g土壤;盲测实验结果显示对于灵敏度范围内的阳性样本均能正确识别,并能正确检测出不同浓度的阳性样本。以FT-fopA片段为靶序列的扩增效率不及基于FT-AKR引物对的扩增。结论：基于FT-AKR片段的引物对扩增效率高,检测土拉弗朗西斯菌具有特异、灵敏的特点,对临床诊断、环境污染监测、防治生物突发事件等具有重要意义。     

转基因棉花MON88701品系特异性实时荧光PCR检测方法的建立

【目的】建立转基因棉花MON88701品系特异性实时荧光聚合酶链反应(polymerase chain reaction,PCR)检测方法。【方法】利用实时荧光PCR技术,根据转基因棉花MON88701品系特异性序列设计引物和探针,建立转基因棉花MON88701实时荧光PCR检测方法,并测定本方法的灵敏度、特异性及可重复性。【结果】建立的检测方法特异于转基因棉花MON88701成分的检测,灵敏度测试表明其定量下限为34拷贝;重复性试验显示其相对标准偏差在可接受范围内。【结论】本研究建立的转基因棉花MON88701品系特异性实时荧光PCR检测方法具有良好的特异性和高灵敏度,适合对转基因棉花MON88701品系进行检测。     

添加有扩增内标的沙门氏菌荧光定量PCR 检测体系的建立与评价

【目的】建立添加有扩增内标(IAC,Internal amplification control)的沙门氏菌EvaGreen荧光定量PCR检测体系,提高PCR检测可靠性。【方法】通过比较已有沙门氏菌属细菌的基因组序列,筛选沙门氏菌属特异检测靶点,设计特异引物;再用复合引物法构建扩增内标,优化参数,建立沙门氏菌内标PCR检测体系,利用特异性和灵敏度实验评价体系的检测性能。【结果】筛选得到的新特异靶点基因编码III型分泌系统蛋白(ssaQ)。针对该基因设计特异引物(SsaQ6),建立了添加有扩增内标的常规PCR和EvaGreen荧光定量PCR检测体系;二者对151株沙门氏菌和34株非沙门氏菌的检测符合率均达100%,对基因组DNA的检测下限达14.9拷贝/PCR和2.76拷贝/PCR;人工污染牛奶样品(初始染菌量:4-6 cfu/10 mL),増菌10 h和8 h后分别可检出沙门氏菌。【结论】本研究发掘的新靶点基因ssaQ特异性强,基于这一新靶点建立的添加有扩增内标的EvaGreen荧光定量PCR比常规内标PCR的检测限更低,重复性更好,快速方便,在12 h内即可得出检测结果,并且定量准确,有利于推进沙门氏菌PCR检测方法的标准化应用。     

添加有扩增内标的副溶血弧菌PCR检测方法的建立

摘要：【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌（Vibrio parahaemolyticus）基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物（vp1332L/vp1332R）,同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343 bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499 bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102 cfu/mL。人工污染实验表明,起始染菌量为1.24 cfu/25 g样品时经8 h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。     

添加有扩增内标的副溶血弧菌PCR检测方法

【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌(Vibrio parahaemolyticus)基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物(vp1332L/vp1332R),同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102cfu/mL。人工污染实验表明,起始染菌量为1.24cfu/25g样品时经8h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。     

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。     

卫氏并殖吸虫PCR和实时荧光PCR快速检测方法的建立

目的建立快速、灵敏、特异的分子生物学检测卫氏并殖吸虫的方法。方法根据卫氏并殖吸虫的特异性基因序列,设计适合于PCR检测的特异性引物及实时荧光PCR特异性引物和探针,并进行灵敏性和特异性试验。结果设计的引物和探针特异性强,所建立的检测方法灵敏度高。应用实时荧光PCR方法的检测灵敏度可达到0.1 pg/μL,比PCR方法的灵敏度高三个数量级。结论本研究所建立的PCR和实时荧光PCR技术检测卫氏并殖吸虫方法的特异性强,灵敏度高,为卫氏并殖吸虫感染的诊治提供了快速的检测技术手段。     

Molecular Detection of <Emphasis Type="Italic">Penicillium griseofulvum</Emphasis> as the Coastal Pollution Indicator

Molecular methods were carried out to detect Penicillium griseofulvum, a dominant species related to heavy metal pollution, which was screened from marine contaminated sediments. Based on differences in internal transcribed spacer (ITS) sequences of Penicillium genus and specific isoamyl alcohol oxidase (IAO) sequences, species-specific primers AS1/RS4 and IAO1/IAO2 of Penicillium griseofulvum were designed and synthesized which were then employed in optimized PCR systems. The detection sensitivities were compared through ordinary PCR and nested-PCR using two pairs of primers, respectively. Both primer pairs could exclusively amplify destined DNA fragment from contaminated environmental samples in our researches. As for primers AS1/RS4, the detection sensitivity for spores (pure spore DNA) could be 10 fg/μl and 10 spores, respectively, and the detection sensitivity for the sediments was 10² spores/0.25 g sediments. While the detection sensitivity of IAO1/IAO2 primers was lower than that of AS1/RS4. Despite the difference in detection sensitivity, it is feasible that the species-specific primers could be used as probes for the detection of environmental pollution dominant species, Penicillium griseofulvum, since the frequency of occurrence and amount of this strain could preferably indicate the pollution degree.     

Penicillium griseofulvum F1959, high-production strain of pyripyropene a, specific inhibitor of acyl-CoA: cholesterol acyltransferase 2

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes cholesterol esterification and plays an important role in the intestinal absorption of cholesterol, hepatic production of lipoproteins, and accumulation of cholesteryl ester within cells. During the course of screening to find ACAT inhibitors from microbial sources, the present authors isolated pyripyropene A from Penicillium griseofulvum F1959. Pyripyropene A, an ACAT2-specific inhibitor, has already been produced from Aspergillus fumigatus. Yet, Aspergillus fumigatus is a pathogen and only produces a limited amount of pyripyropene A, making the isolation of pyripyropene A troublesome. In contrast, Penicillium griseofulvum F1959 was found to produce approximately 28 times more pyripyropene A than Aspergillus fumigatus, plus this report also describes the ideal conditions for the production of pyripyropene A by Penicillium griseofulvum F1959 and its subsequent purification.     

Isolation and structure of SCH 351633: a novel hepatitis C virus (HCV) NS3 protease inhibitor from the fungus Penicillium griseofulvum.

A new hepatitis C virus (HCV) protease inhibitor designated as Sch 351633 (1) was isolated from the fungus, Penicillium griseofulvum. Structure elucidation of 1 was accomplished by analysis of spectroscopic data, which determined compound 1 to be a bicyclic hemiketal lactone. Compound 1 exhibited inhibitory activity in the HCV protease assay with an IC50 value of 3.8 microg/mL.     

Cyclopiazonic acid production by submerged cultures of Penicillium and Aspergillus strains

Abstract 62 isolates of Penicillium and Aspergillus were screened for cyclopiazonic acid (CPA) production by surface and submerged culture on different media. The production of this mycotoxin was restricted to Penicillium camembertii group II (and its domesticated form P. camembertii ), P. griseofulvum , and Aspergillus flavus (and its domesticated form A. oryzae ). The best yield of CPA was obtained by a strain of P. griseofulvum , but several strains of P. camembertii group II were also good producers. Propionic acid (500 and 1000 mg/l medium) did not enhance the production of CPA. The best yields of CPA were obtained in submerged culture, but in some cases growth and CPA production only occured in surface culture. A simplified procedure for isolation of CPA is described.     

Metabolic synergies in the biotransformation of organic and metallic toxic compounds by a saprotrophic soil fungus

Applied Microbiology and Biotechnology - The saprotrophic fungus Penicillium griseofulvum was chosen as model organism to study responses to a mixture of hexachlorocyclohexane (HCH) isomers...     

Effect of 3-phenylpropanoic Acid on growth of and cellulose utilization by cellulolytic ruminal bacteria

Patulin production by Penicillium griseofulvum was monitored with Sep-Pak cartridges and high-pressure liquid chromatography. Determination and quantification of this metabolite proved to be very simple, and our method saved time and a large amount of organic solvents.     

一株扩展青霉拮抗菌的分离、鉴定及抑菌活性物质

【目的】筛选有效抑制扩展青霉(Penicillium expansum)的拮抗菌,并鉴定其所产抑菌物质的主要种类及相对含量。【方法】从苹果表面分离到拮抗扩展青霉的菌株BA-16,经形态学、生理生化及16S r RNA基因序列分析对该菌进行鉴定;根据已知脂肽类抗生素合成相关基因序列设计3对特异性引物对菌株BA-16进行检测,对PCR产物克隆、测序和BLAST分析,采用酸沉淀法从菌株发酵液中制备出抑菌物质粗提液,对活性粗提物进行HPLC和MALDI-TOF-MS分析。【结果】经鉴定,BA-16被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),所得PCR产物经测序和BLAST分析,证实BA-16带有sfp和fen B基因。HPLC和MS结果显示菌株发酵液中含有Fengycin和Surfactin两种脂肽类产物,Fengycin是拮抗扩展青霉的主要因素。【结论】本研究对于苹果采后青霉病的生物防治具有良好的应用开发前景。     

Bioremediation of aflatoxins by some reference fungal strains.

Aspergillus parasiticus RCMB 002001 (2) producing four types of aflatoxins B1, B2, G1, and G2 was used in this study as an aflatoxin-producer. Penicillium griseofulvum, P. urticae, Paecilomyces lilacinus, Trichoderma viride, Candida utilis, Saccharomyces cerevisiae as well as a non-toxigenic strain of Aspergillus flavus were found to be able to exhibit growth on aflatoxin B1-containing medium up to a concentration of 500 ppb. It was also found that several fungal strains exhibited the growth in co-culture with A. parasiticus, natural aflatoxins producer, and were able to decreased the total aflatoxin concentration, resulting in the highest inhibition percentage of 67.2% by T viride, followed by P. lilacinus, P. griseofulvum, S. cerevisiae, C. utilis, P. urticae, Rhizopus nigricans and Mucor rouxii with total aflatoxin inhibition percentage of 53.9, 52.4, 52, 51.7, 44, 38.2 and 35.4%, respectively. The separation of bioremediation products using GC/MS revealed that the toxins were degraded into furan moieties.     

Production of penicillin by fungi growing on food products: identification of a complete penicillin gene cluster in Penicillium griseofulvum and a truncated cluster in Penicillium verrucosum

Mycobiota growing on food is often beneficial for the ripening and development of the specific flavor characteristics of the product, but it can also be harmful due to the production of undesirable compounds such as mycotoxins or antibiotics. Some of the fungi most frequently isolated from fermented and cured meat products such as Penicillium chrysogenum and Penicillium nalgiovense are known penicillin producers; the latter has been shown to be able to produce penicillin when growing on the surface of meat products and secrete it to the medium. The presence of penicillin in food must be avoided, since it can lead to allergic reactions and the arising of penicillin resistance in human-pathogenic bacteria. In this article we describe a study of the penicillin production ability among fungi of the genus Penicillium that are used as starters for cheese and meat products or that are frequently isolated from food products. Penicillium griseofulvum was found to be a new penicillin producer and to have a penicillin gene cluster similar to that of Penicillium chrysogenum. No other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except P. verrucosum, which contains the pcbAB gene (as shown by hybridization and PCR cloning of fragments of the gene) but lacks pcbC and penDE. Antibacterial activities due to the production of secondary metabolites other than penicillin were observed in some fungi.     

Some properties of free and immobilized alpha-amylase from Penicillium griseofulvum by solid state fermentation

Alpha-amylase was produced from Penicillium griseofulvum by an SSF technique. Alpha-amylase was immobilized on Celite by an adsorption method. Various parameters, such as effect of pH and temperature, substrate concentration, operational and storage stability, ability to hydrolyze starch and products of hydrolysis were investigated; these findings were compared with the free enzyme. The activity yield of immobilization was 87.6%. The optimum pH and temperature for both enzymes were 5.5 degrees C and 40 degrees C, respectively. The thermal, and the operational and storage stabilities of immobilized enzyme were better than that of the free enzyme. Km and Vmax were calculated from Lineweaver-Burk plots for both enzymes. Km values were 9.1 mg mL(-1) for free enzyme, and 7.1 mg mL(-1) for immobilized enzyme. The Vmax of the immobilized enzyme was approximately 40% smaller than that of the free enzyme. The hydrolysis ability of the free and immobilized enzyme were determined as 99.3% and 97.9%, respectively. Hydrolysis products of the a-amylase from P. griseofulvum were maltose, unidentified oligosaccharides, and glucose.