Prevention of Chinese Sacbrood Virus Infection in Apis cerana using RNA Interference

Chinese sacbrood virus (CSBV) is the pathogen of Chinese sacbrood disease, which poses a serious threat to honeybee Apis cerana, and tends to cause bee colony and even the whole apiary collapse. Here we report on prevention of CSBV infection by feeding second instar larvae of A. cerana with specific sequences of CSBV double-stranded RNA (dsRNA). Protection of the bee larvae from CSBV by ingestion of CSBV-derived dsRNA was further demonstrated by quantitative real-time PCR (qRT-PCR) and northern blot analysis. The result provides a potential method to protect A. cerana from CSBV infection.     

Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.     

RNAi靶向VP2基因抑制中蜂囊状幼虫病毒在中华蜜蜂幼虫体内复制研究

中蜂囊状幼虫病(Chinese sacbrood disease,CSBD)是造成中华蜜蜂患病死亡的主要原因之一,目前尚无有效的治疗方法。为了研究靶向中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)结构蛋白VP2基因的siRNA介导的RNA干扰(RNA interference,RNAi)作用和其对CSBV在中华蜜蜂幼虫体内复制的影响,设计合成针对CSBV VP2基因的特异性siRNA,以100 nM的浓度与pEGFPN1-VP2-CSBV融合表达载体共同转染至293T细胞中,通过荧光显微镜和流式细胞仪观察和分析siRNA在体外对CSBV VP2基因表达的干扰效果。同时,将siRNA(1μg/μL)和1×10~7拷贝数的CSBV共同饲喂2日龄中华蜜蜂幼虫,检测幼虫体内CSBV拷贝数和幼虫存活率,研究siR-NA对中华蜜蜂幼虫体内CSBV复制的影响。荧光结果显示,在293T细胞中siRNA能抑制CSBV VP2蛋白的表达,并且通过流式细胞仪检测分析发现干扰效果接近40%。幼虫饲喂实验表明,饲喂siRNA组在各时间点幼虫体内CSBV拷贝数均低于CSBV对照组,且在摄入siRNA后感染CSBV的幼虫存活率明显上升,与CSBV组差异极显著(P<0.01)。通过本研究,证明了针对CSBV结构蛋白VP2基因的特异性siRNA能够介导产生RNAi,影响CSBV在中蜂体内的复制,为深入研究CSBV VP2基因的功能和研发抗CSBV生物制剂提供了理论基础。     

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。     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Characterization of sacbrood virus and its infection in Apis cerana in the Qiandao Lake region in Zhejiang Province,China

Chinese sacbrood virus (CSBV) is a disastrous virus that fatally threatens honeybees (Apis cerana) in China. Here, we identified the complete genome of CSBV using the rapid amplification of cDNA ends (RACE) method and compared the obtained sequences with those of other SBVs through multiple sequence alignment. The results showed that the full-length of CSBV genome from Zhejiang Province (CSBV-ZJ) is 8824 bp and contains a single, large open reading frame (ORF) that encodes 2842 amino acids flanked by 190 nucleotides (nts) of the 5′-untranslated region (UTR) and 108 nts of the 3′-UTR. The multiple genome comparison showed that SBVs have five conserved domains and that CSBV-ZJ belong to SBV type II. The phylogenetic analysis demonstrated that the CSBV-ZJ strain was similar to the CSBV from Fuzhou (CSBV-FZ) strain. Colonies collected from most of the sampling sites in the area surrounding Qiandao Lake were carriers of CSBV. The haplotypes of the VP1 gene formed a network with radiated and reticular structures. In conclusion, this characterization of the CSBV-ZJ genome will help us understand the SBV prevalence mechanism and aid the protection of honeybees.     

O1群霍乱弧菌实时荧光定量PCR快速检测方法的建立

目的:建立针对O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,并进行模拟粪便标本的检测评价。方法:根据O1群霍乱弧菌O抗原编码基因rfb的特异性序列设计引物和TaqMan探针,建立检测O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,对所建立的方法分别进行实验室内的灵敏度及特异性评价;将O1群霍乱弧菌灭活菌株悬液倍比稀释后与健康成人新鲜粪便混匀,制备成模拟带菌者粪便标本,提取DNA,进行Taq-Man PCR检测,用以评价该方法。结果:建立了快速检测O1群霍乱弧菌的实时荧光定量TaqMan PCR方法,灵敏度为每反应体系104拷贝;该方法对其他14种肠道菌DNA没有扩增;该方法对模拟粪便标本的检测灵敏度为每反应体系102 CFU。结论:建立了一种快速、高效检测O1群霍乱弧菌的荧光定量PCR检测方法,该方法可用于O1群霍乱弧菌临床粪便标本的检测。     

WSSV和IHHNV二重实时荧光PCR检测方法的建立

根据基因库中对虾白斑综合征病毒WSSV(AF369029)和传染性皮下及造血器官坏死病毒IHHNV(AF218226)基因序列,设计了WSSV和IHHNV的两对特异性引物和两条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测WSSV和IHHNV的二重实时荧光PCR方法。该方法特异性好,对WSSV和IHHNV的检测敏感性分别达到2和20个模板拷贝数;此外抗干扰能力强,对WSSV和IHHNV不同模板浓度进行组合,仍可有效地同时检测这二个病毒。对保存的30份经常规PCR检测仅为WSSV或IHHNV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中1份为WSSV和IHHNV混合感染。本研究建立的二重实时荧光PCR方法用于WSSV和IHHNV的检测具有特异、敏感、快速、定量等优点。     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。     

TaqMan—MGB荧光定量RT-PCR技术快速检测H5亚型禽流感病毒

建立以TaqMan-MGB荧光探针为特点的荧光定量RT-PCR方法,用于检测H5亚型禽流感病毒。针对H5亚型禽流感病毒血凝素(HA)基因保守区域设计特异性引物与TaqMan-MGB荧光探针,筛选并优化荧光定量RT-PCR反应体系与反应条件,用以提高方法的特异性、敏感性与准确性;并通过体外克隆技术建立病毒基因拷贝数进行定量分析。结果表明:引物与探针的优化浓度分别640nmol/L和480nmol/L,体系具有良好的保守性和特异性,与其他呼吸道病毒均无交叉反应。方法检测灵敏度为100拷贝/反应,标准曲线线性范围为107～102拷贝/反应,从病毒核酸提取至检测完成仅需3h左右,操作简便,重现性好。本研究建立的TaqMan-MGB荧光定量PCR方法特异、敏感、快速,适合于临床实验室进行H5亚型禽流感病毒的快速定量检测。     

Rapid detection and quantification of Prorocentrum minimum by loop-mediated isothermal amplification and real-time fluorescence quantitative PCR

The dinoflagellate Prorocentrum minimum was successfully detected using loop-mediated isothermal amplification (LAMP) and real-time fluorescence quantitative PCR (RTFQ-PCR). Both specificity and sensitivity testing in the two methods have been validated. In the LAMP assay, the specific ladder-like pattern of bands only appeared in those templates containing P. minimum. The sensitivity of LAMP was tenfold higher than conventional PCR. In RTFQ-PCR assay, only positive amplifications were detected from those samples containing P. minimum. RTFQ-PCR can detect 0.1 cells and 10 pg of DNA within 40 cycles, showing its high sensitivity. Cells could be quantified according to standard curves in agreement with the quantification by standard microscopy counting methods. The LAMP method therefore is appropriate for on-the-spot testing because of its rapidity and simplification, and the RTFQ-PCR is fit for laboratory testing owing to its accurate quantification. The two methods are of significance in forecasting red tides.     

Three-dimensional structure of the Chinese Sacbrood bee virus

The RNA of Chinese Sacbrood Bee Virus (CSBV) was purified and used as template to obtain a 1096 bp cDNA fragment by RT-PCR amplification. This DNA fragment was cloned into pGEM-T Easy Vector for sequencing. Analyses of the sequenced CSBV RNA fragment revealed a nucleotide sequence homology of 87.6% and a deduced amino-acid sequence homology of 94.6% with that of the Sacbrood Virus (SBV), indicating that CSBV is a different but highly homologous virus of SBV. The three-dimensional (3D) structure of CSBV was determined at 2.5 nm resolution by using electron cryo-microscopy (cryoEM) and computer reconstruction methods. The 3-D structure showed that the capsid has aT = 1 (orP = 3) icosahedral capsid shell with a smooth surface. There were 12 pentons at its icosahedral vertices (5-fold axes) and 132 holes penetrating the shell. The 3-D structure also revealed densities corresponding to the CSBV genome, suggesting icosahedrally-ordered RNA organization, a novel feature not previously reported for any picornaviruses.     

Evaluation of a duplex real-time PCR assay to detect MRSA from broth culture,human sera seeded with MRSA and from patient's serum

The need for rapid methods in order to precisely detect methicillin-resistant Staphylococcus aureus (MRSA) is extensively acknowledged. This study evaluated a quantitative real-time PCR assay targeting mecA (encoding high level resistance to methicillin) and femB (a specific genomic marker for S. aureus) genes to detect MRSA from broth culture, from serum seeded with MRSA and straight from the patient''s serum. One hundred and thirty-five clinical isolates of MRSA strains and different species were utilised in this study. In addition, a pilot study with 9 patients'' serum samples was performed. The sensitivity and specificity values for this assay were 99% and 100% respectively. The detection limit for this method was 1.23×10² CFU/ml from the serum seeded with MRSA cells and the limiting concentration of DNA for detection was 18 fg, which equates to 5.14 genomic DNA copies. In addition, this assay detected MRSA from patient''s serum (7 out of 9) with sensitivity of 77.8%. Overall, the assay was rapid, efficient, sensitive and easy to perform.     

Genetic and phylogenetic analysis of the honey bee sacbrood virus from jiangxi isolates

The high prevalence of honeybee viral diseases poses a severe threat to the health of honeybees and causes substantial economic losses worldwide. Sacbrood virus (SBV) is a single-strand RNA virus that infects honeybees at all life stages. The infection can shorten the lifespan of adult bees and is lethal to larvae. SBV is the major cause of honeybee losses in Asia. Genetic and phylogenetic analyses of SBV isolates from different areas have been previously conducted. However, the impact of Apis mellifera Linnaeus and Apis cerana Fabricius coexistence on the infection and phylogeny of SBV remains unknown. In this study, we collected A. cerana and A. mellifera samples from commercial apiaries, only A. cerana in mountainous region. SBV prevalence was evaluated in three commercial apiaries of Jinxi, Tonggu and Nanchang and two mountainous regions of Zixi and Yifeng. In our sampling location, we found a higher SBV prevalence in the mountainous regions than in commercial apiaries. Partial structural polyprotein coding sequences were sequenced and compared with other GenBank SBV isolates. Phylogenetic tree topologies showed that SBV isolates form two major groups based on their host specificity, and isolates from same country tend to cluster together in subclades, indicating that the host and geographic region has significant effects on SBV strain specificity.     

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5–3′ exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R² > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC–MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation.     

LightCycler real-time PCR for rapid detection and quantitation of Mycobacterium leprae in skin specimens

Diagnosis of leprosy is usually based on clinical features and skin smear results including the number of skin lesions. Mycobacterium leprae is not cultivable and bacterial enumeration by microscopic examination is required for leprosy classification, choice in choosing and monitoring chemotherapy regimens, and diagnosis of relapse. However, detection and quantification using standard microscopy yields results of limited specificity and sensitivity. We describe an extremely sensitive and specific assay for the detection and quantification of M. leprae in skin biopsy specimens. Primers that amplified a specific 171-bp fragment of M. leprae 16S rRNA gene were chosen and specificity was verified by amplicon melting temperature. The method is sensitive enough to detect as low as 20 fg of M. leprae DNA, equivalent to four bacilli. The assay showed 100% concordance with clinical diagnosis in cases of multibacillary patients, and 50% of paucibacillary leprosy. The entire procedure of DNA extraction and PCR could be performed in c. 3 h. According to normalized quantitative real-time PCR, the patients in this study had bacilli numbers in the range of 1.07 x 10(2) -1.65 x 10(8) per 6-mm3 skin biopsy specimen. This simple real-time PCR assay is a facile tool with possible applications for rapid detection and simultaneous quantification of leprosy bacilli in clinical samples.     

Development and Evaluation of Methods To Detect Nucleopolyhedroviruses in Larvae of the Douglas-Fir Tussock Moth, Orgyia pseudotsugata (McDunnough)

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r² = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.     

中华蜜蜂幼虫原代细胞培养及中华蜜蜂囊状幼虫病毒在培养的细胞中的复制

【目的】建立一种适用于蜜蜂源的细胞培养方法,为蜜蜂源细胞培养和蜂病毒的研究奠定基础。【方法】比较在Grace和WH2两种昆虫细胞培养基中培养的中华蜜蜂 Apis cerana cerana 幼虫原代细胞状况,筛选出适用于中华蜜蜂幼虫原代细胞培养的最佳培养基,并通过细胞活力比较,确定用于中华蜜蜂幼虫原代细胞培养的适宜胎牛血清(fetal bovine serum, FBS)浓度。在此基础上,用该原代细胞接种中华蜜蜂囊状幼虫病毒（Chinese sacbrood bee disease, CSBV）,并通过实时定量RT-PCR方法对病毒复制情况进行检测。【结果】相对于WH2培养基,在Grace培养基中生长的细胞大而圆,透明,边缘整齐,无颗粒物,活力明显高于WH2培养,且含15% FBS的Grace培养基更适合于中华蜜蜂幼虫原代细胞培养。CSBV接种在该原代细胞,能够复制增殖,同时伴随宿主细胞快速分裂。【结论】中华蜜蜂幼虫原代细胞培养基在含15%FBS的Grace中能够良好生长,并且CSBV可以在该原代细胞中进行复制。