Application of Abnormal Touchdown PCR with High Degeneracy Primer in Amplification of Large-Family Genes

[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.

Use of species-specific PCR for the identification of 10 sea cucumber species

We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species.Ten reverse species-specific primers designed from the 16 S rRNA gene,in combination with one forward universal primer,generated PCR fragments of ca.270 bp length for each species.The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species.Amplification was observed in specific species only.The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber,and was proven to be a useful,rapid,and low-cost technique to identify the origin of the sea cucumber product.

The Frequency of the v-AKT Murine Thymoma Viral Oncogene Homologue 1 Gene Amplification among Sudanese Women with Ovarian Cancer: A Cross-Sectional Study

Background: Protein kinase B (AKT/PKB) family is frequently amplified in ovarian cancer (OC). To the greatest of our knowledge, there is a lack of published reports about the amplification of the genes belonging to the AKT family among Sudanese women with OC. The present study was conducted to detect the AKT1 gene amplification and its association with tumour types, grades, and ages among Sudanese women with OC, bearing in mind the ethnic variation. Methods: This institution-based study included 79 cases of women diagnosed with ovarian cancer (OC) at Omdurman Maternity Hospital in the period 2013-2018. Formalin-fixed, paraffin-embedded (FFPE) tissue sections were used to extract RNA. AKT1 gene amplification was assessed using quantitative real-time PCR. Results: The mean age (±SD) of included women was 49.29 (±13.612). The amplification of AKT1 gene was observed in 18/79 (22.8%) of OC women, with a high frequency in women with undifferentiated 1/2 (50%), clear cell 2/6 (33.3%), mucinous 3/11 (27.3%), endometrioid 3/17 (17.6%), and serous carcinomas 5/30 OC (16.7%). High frequency was seen in women with low (26.3%;n = 10/28) rather than in higher (19.5%;n = 8/33) grade carcinoma, and in older (25.8%;n = 8/23) rather than younger (18.2%;n = 2/9) women. No significant association between AKT1 gene amplification and tumour types, grades, and ages of women was observed (Fisher’s Exact test: p = 0.405, 0.593 and 0.851, respectively). Conclusion: AKT1 gene amplification arises in around one-fifth of Sudanese women with ovarian cancer (OC). It is seen more in undifferentiated, clear cell, and mucinous tumours types, and more frequently in low tumour grade and older women, but not to a statistically significant level. These outcomes sustenance previous studies suggesting that activated AKT genes have a vital role in OC progression and may offer a plan for targeted therapy and prognostic evaluation.

Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China

Botryosphaeriaceae species are important causal agents of blueberry stem blight worldwide. Blueberry stem blight has become an important disease, potentially affecting the quality and production of blueberries in China. It is difficult and time-consuming to identify at the species level using morphological methods. The aim of this study was to develop polymerase chain reaction（PCR） assays for the diagnosis and early detection of latent infections of blueberry stems by Botryosphaeria spp. Species-specific primers, based on the ribosomal DNA internal transcribed spacer region and β-tubulin gene, were designed and selected for use in PCR assays. Three primer pairs, Lt347-F/R for Lasiodiplodia theobromae, Np304-F/R for Neofusicoccum parvum and FaF/Bt2b for Botryosphaeria dothidea, successfully amplified specific PCR fragments of different sizes on pure cultures or from blueberry stems inoculated and naturally infected blueberry plants with three pathogens, respectively. These primers did not amplify any PCR fragments from other blueberry stem disease-associated pathogens, such as Phomopsis spp. and Pestalotiopsis spp. This PCR protocol could detect as low as 1 00 pg to 1 ng of purified fungal DNA. This PCR-based protocol could be used for the diagnosis and detection of these pathogens from pure cultures or from infected blueberry plants.

旋转式三温区微流控PCR扩增平台的研制

微流控芯片用于聚合酶链式反应(PCR)可提高检测效率和自动化程度,但目前把核酸提取、扩增、检测功能集成到一块芯片上仍比较困难,此外,微流控芯片专用PCR仪也存在温度控制不够快速、精准的问题。作者采用空间上温度循环代替时间上温度循环的设计思路,搭建了一套旋转式三温区微流控PCR扩增平台,对大肠杆菌进行核酸提取和扩增。结果表明,旋转式三温区微流控PCR扩增平台拥有较好的热均匀性和热稳定性,平台的升降温速率分别为3.5℃/s和2.67℃/s,单次循环时间为110 s。与9700型PCR仪相比,所建平台的温度控制方案更为简单、升降温速率更高、循环时间更短。本研究结果可为实现核酸提取、扩增一体化的微流控PCR设备的研发提供参考。

一种基于real-time PCR技术的TTV检测方法的建立及应用

目的:本研究旨在开发一种具有更高灵敏度和特异性的TTV检测技术,为揭示TTV在多种疾病过程中的作用提供重要的技术支持。方法:为了更精确、灵敏的检测TTV,本研究分析了目前公布的所有亚型的TTV基因序列,在此基础上建立了一种基于UTR区域的real-time PCR检测方法,并与文献报道应用较为广泛的PCR检测方法进行了对比。结果:本研究建立的方法在1×10^(7)~1×10^(1) copies/μL标准品浓度范围内具有良好的线性关系,相关系数为1.000,斜率为-3.446,检测下限为1×10^(1) copies/μL。重复性试验结果显示,组内变异系数为7.22%,表明本方法重复性、稳定性较强。针对30份临床样本,使用本研究建立的real-time PCR检测方法及目前被多个研究所使用的4套引物进行对比。结果表明,本研究所建立的方法灵敏度显著高于文献中报道的4种方法(P<0.01);Sanger测序结果表明,本方法检测出的30份阳性样本均为TTV,检测特异性为100%。结论:本研究采用基于TaqMan探针的real-time PCR检测方法,检测灵敏性高、覆盖基因型范围广,尤其对于TTV病毒载量较低的情况下能够进行定量检测,对于TTV病毒的致病性及作为免疫标志物的应用提供重要的技术支持。

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

Rapid Amplification of 5′ cDNA End of S. Liaotungensis Choline Monooxygenase Using Inverse PCR RACE

Based on part of a known cDNA sequence of Suaeda Liaotungensis choline monooxygenase, the authors successfully cloned the 5′ cDNA end of Suaeda Lianotungensis choline monooxygenase using Inverse PCR RACE with a specially designed 5′-phosphated RT primer and two pairs of specific inverse PCR primers. Compared with the anchored PCR RACE, inverse PCR RACE has better specificity and higher amplification.

Identification of various Biomphalaria alexandrina strains collected from five Egyptian governorates using RAPD and species-specific PCR techniques

The first generation of Biomphalaria snails collected from five Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta) were sub-jected to species-specific PCR assays and the results showed that snails collected from the field were B. alexandrina, and there was no evidence for the pres-ence of B. glabrata. The snails were subjected also to RAPD- PCR technique. The results showed that dif-ferent fingerprints with each B. alexandrina strain were produced with varying numbers of bands rang-ing in size from 123.6 to 796.6 bp depending on the snail strain and the primer used. Many specific bands were obtained with the four primers in each strain. Primer OPA-1 amplified the highest number of spe-cific bands (26 bands) and gave the highest poly-morphism among the primers used (100% polymor-phism). The estimated similarity coefficients among B. alexandrina strains based on the RAPD-PCR pro-files ranged from 0.56 to 0.72. The highest similarity coefficient (0.72) was recorded between the strains of Ismailia and Kafr El-Sheikh, while the lowest coeffi-cient (0.56) was reported between the strains of SPSC and Ismailia.

药品中金黄色葡萄球菌实时荧光定量PCR精准快检

为实现药品中金黄色葡萄球菌的精准快速检测,通过加入内参(internal amplification control,IAC),优化PCR反应体系,建立能够实时监控过程的基于内参的金黄色葡萄球菌实时荧光定量PCR快检方法,并对方法的特异性、检测限、重现性、可行性进行验证。验证结果表明,所建立方法的特异性良好,仅金黄色葡萄球菌呈现典型扩增曲线;对金黄色葡萄球菌基因组DNA的检测限为0.23 pg/μL;C_(t)值与模板拷贝数呈现出良好的线性关系(R^(2)=0.99);重现性实验的相对标准偏差均小于3.0%;人工污染药品增菌10 h后即可检出金黄色葡萄球菌。该方法不仅缩短了药品中金黄色葡萄球菌的检验时间,还可以实时监测PCR反应过程,有效防止“假阴性”结果的发生,能够作为确认金黄色葡萄球菌的补充方法。

Advances in Application of PCR Amplification in Etiologic Diagnosis

In recent years,with the rapid development of molecular biology diagnostic technology,many new polymerase chain reaction(PCR)technologies with high specificity and good sensitivity have gradually been developed.While expanding the range of detection methods,these technologies inevitably have some disadvantages.Therefore,in clinical pathogen diagnosis,medical personnel should choose the detection method according to the detection purpose and pathogen characteristics.In this paper,the basic principle,application scope,advantages and disadvantages and development of various emerging PCR diagnostic techniques are respectively described in order to provide a theoretical reference for the selection of pathogenic biological diagnostic techniques in the clinical practice.

诺如病毒TaqMan荧光定量PCR检测方法的设计与优化

为设计一种快速、高灵敏度检测诺如病毒(Norovirus,NV)的方法.本文通过检索参考NCBI数据库中收录的国内典型流行的NV毒株BJSMQ的基因序列NC_039476.1,合成了NV的标准重组质粒,根据NV的ORF1基因保守序列设计一组特异性引物和荧光探针;通过正交试验进行程序及试剂反应体系的优化,设计TaqMan荧光定量PCR检测方法.结果表明该检测方法具有高灵敏度,最低检测限可达1.2 copies/μL,且在标准重组质粒浓度1.2×10^(5)~1.2×10^(0) copies/μL范围内具有较好的线性关系,其R^(2)=0.9907;该方法特异性强,在病毒的混合重组质粒样液中仅与NV病毒反应;组内和组间的重复性良好,变异系数(CV)值均小于2%;在数字PCR试验中进一步验证了该方法的可行性.本文设计的TaqMan荧光定量PCR检测方法可用于NV毒株的快速准确检测,有望成为NV诊断的有效工具.

SAT-TB联合FQ-PCR检测在痰涂片阴性肺结核诊断中的效能

目的:分析RNA恒温扩增实时检测技术(SAT-TB)联合荧光定量聚合酶链反应(FQ-PCR)检测在痰涂片阴性肺结核患者诊断中的效能。方法:选取2020年7月至2022年7月该院收治的75例疑似痰涂片阴性肺结核患者进行前瞻性研究。采集所有患者肺泡灌洗液标本,采用SAT-TB、FQ-PCR法进行检测,以结核分枝杆菌痰培养结果为“金标准”,比较SAT-TB、FQ-PCR单项及联合检测在痰涂片阴性肺结核诊断中的效能。结果:金标准结果显示,75例疑似痰涂片阴性肺结核患者中,阳性48例,阴性27例;SAT-TB检测结果显示,阳性36例,阴性39例;FQ-PCR检测结果显示,阳性37例,阴性38例;SAT-TB联合FQ-PCR检测结果显示,阳性47例,阴性28例;SAT-TB联合FQ-PCR检测诊断痰涂片阴性肺结核的灵敏度、准确度均高于SAT-TB、FQ-PCR单项检测诊断,漏诊率低于SAT-TB、FQ-PCR单项检测诊断,差异有统计学意义(P<0.05)。结论:SAT-TB联合FQ-PCR检测诊断痰涂片阴性肺结核的效能高于二者单项检测诊断效能。

Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.

大豆根瘤菌菌株5873 PCR快速检测方法的建立及应用效果评价

【目的】大豆根瘤菌(Bradyrhizobium japonicum)是微生物肥料重要的功能菌种之一,可通过生物固氮为大豆生长提供氮素,实现节本增效,菌株5873作为其典型代表,已广泛应用于农业生产。筛选并鉴定其特异引物,建立大豆根瘤菌5873菌株水平的快速检测方法,对微生物肥料生产菌种鉴定、产品质量检测和功能评价至关重要。【方法】以商业化菌株大豆根瘤菌5873为供试材料,基于其全基因组序列和NCBI数据库中大豆根瘤菌种内参比菌株相关序列,以及与其高度同源(基因组ANI值大于99.95%)的大豆根瘤菌USDA 6T差异片段,通过多重序列比对,进行特异引物设计和筛选,获得特异性引物对,并通过对PCR反应条件/体系优化、特异性及灵敏度检测,建立大豆根瘤菌5873的快速检测方法。然后,采用盆栽试验,将大豆根瘤菌5873与其他根瘤菌菌株混合接种于大豆根际,应用上述方法对大豆根瘤菌5873竞争结瘤能力进行评价和方法验证。【结果】筛选获得了一组特异引物4-4和Q1(4-4-F 5′-GATAAGGCCACGGGTGAACA-3′/4-4-R 5′-CACTCGATAAGCTCCGCTGT-3′和Q1-F 5′-CCGGTCGTGACTGGAATGAT-3′/Q1-R 5′-TCGAGGCCTACAAGAACGTC-3′),优化并建立了PCR快速检测方法,即反应体系:Premix TaqTM 12.5μL,引物各1.0μL,基因组DNA 15 ng左右,加ddH2O补足至25μL;反应条件:95℃预热5 min,94℃变性45 s,61℃退火45 s,72℃延伸60 s,30个循环,再72℃延伸10 min。通过凝胶电泳检测目的条带的有无(355和218 bp),即可实现大豆根瘤菌5873的快速检测,该方法检出灵敏度为1850 CFU/μL。此外,借助该方法可以成功地评价大豆根瘤菌5873竞争结瘤能力,与传统BOX-PCR评价结果一致。【结论】大豆根瘤菌5873快速鉴定方法的建立实现了以菌体发酵液或根瘤破碎提取液为模板,直接进行PCR扩增的鉴定操作,省去了根瘤的分离、根瘤菌分离纯化、培养、DNA提取及测序鉴定等繁琐的环节,大大减少了工作量,只需短短几个小时便可准确检测大豆根瘤菌5873,为微生物肥料中根瘤菌菌剂的产品质量检测和竞争结瘤能力评价提供了参考。

Amplification of chromosome 21q22.3 harboring trefoil factor family genes in liver fluke related cholangiocarcinoma is associated with poor prognosis

AIM： To determine allelic imbalance on chromosomal region 21q22-qter including trefoil factor family genes （TFF） in cholangiocarcinoma （CCA） patients and analyze the correlation between allelic imbalances and clinicopathological parameters. METHODS： Quantitative PCR amplification was performed on four microsatellite markers and trefoil factor family genes （TFF1, TFF2, and TFF3） using a standard curve and SYBR Green I dye method. The relative copy number was determined by DNA copy number of tested locus to reference locus. The relative copy number was interpreted as deletion or amplification by comparison with normal reference range. Associations between allelic imbalance and clinicopathological parameters of CCA patients were evaluated by χ^2-tests. Kaplan-Meier method was used to analyze survival. RESULTS： The frequencies of amplification at D21S1890, D21S1893, and TFF3 were 32.5%, 30.0%, and 28.7%, respectively. Patients who had amplification at regions covering D21S1893, D21S1890, and TFF showed poor prognosis, whereas patients who had deletion showed favorable prognosis （mean： 51.7 wk vs 124.82 wk, P = 0.012）. Multivariate Cox regression analysis revealed that amplification of D21S1893, D21S1890 and TFF, blood vessel invasion, and staging were associated with poor prognosis. CONCLUSION： D21S1893-D21S1890 region may harbor candidate genes especially TFF and serine protease family, which might be involved in tumor invasion and metastasis contributing to poor survival. The amplification in this region may be used as a prognostic marker in the treatment of CCA patients.

奶牛乳房炎源肺炎克雷伯菌PCR和LAMP检测方法的建立

为快速鉴定因肺炎克雷伯菌侵染引起的奶牛乳房炎,进一步为该疾病提供更合理的治疗方案,分别以肺炎克雷伯菌16S-23S rDNA内部转录间隔层序列(Internal transcribed spacer,ITS)和脲酶UreD基因为靶点,利用PCR技术和环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)构建奶牛乳房炎源肺炎克雷伯菌快速鉴定方法。结果表明,利用PCR技术扩增ITS序列能准确鉴定出肺炎克雷伯菌,在核酸质量浓度为5×10^(-2)ng/μL的环境中依然能实现稳定扩增;利用LAMP技术扩增UreD基因的最佳反应温度为62℃,其最低检测核酸质量浓度为5×10^(-7)ng/μL,检测灵敏度是PCR方法的105倍。综上,建立了PCR技术和LAMP技术快速鉴定奶牛乳房炎型肺炎克雷伯菌的方法,特异性强且灵敏度高。

Robust and efficient direct multiplex amplification method for large-scale DNA detection of blood samples on FTA cards

Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ionizing radiations (X-rays, γ-rays, alpha particles, etc.) and ultraviolet (UV) radiation lead to the DNA damage. Polymerase chain reaction (PCR) is one of the most wildly used techniques for detecting DNA damage as the amplification stops at the site of the damage. Improvements to enhance the efficiency of PCR are always required and remain a great challenge. Here we establish a multiplex PCR assay system (MPAS) that is served as a robust and efficient method for direct detection of target DNA sequences in genomic DNA. The establishment of the system is performed by adding a combination of PCR enhancers to standard PCR buffer. The performance of MPAS was demonstrated by carrying out the direct PCR amplification on 1.2 mm human blood punch using commercially available primer sets which include multiple primer pairs. The optimized PCR system resulted in high quality genotyping results without any inhibitory effect indicated and led to a full-profile success rate of 98.13%. Our studies demonstrate that the MPAS provides an efficient and robust method for obtaining sensitive, reliable and reproducible PCR results from human blood samples.

Differentiation of Avian Pathogenic <i>Escherichia coli</i>Strains from Broiler Chickens by Multiplex Polymerase Chain Reaction (PCR) and Random Amplified Polymorphic (RAPD) DNA

We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by multiplex PCR and random amplification of polymorphic DNA (RAPD) tests. The multiplex polymerase chain reaction (PCR) which was used as tentative criteria of APEC targets 8 virulence associated genes;enteroaggregative toxin (astA), Type 1 fimbria adhesion (fimH), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. E. coli strains already typed as an APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of an APEC is possession of 5 or more virulence associated genes;we found that all 50 E. coli strains were classified as APEC strains. The RAPD analysis showed that the E. coli strains could be grouped into 35 of RAPD types by using these two different RAPD primer sets, RAPD analysis primer 4 5'AAGAGCCCGT5', and RAPD analysis primer 6 5'CCCGTCAGCA3'. The current study confirmed the endemic nature of APEC in broiler flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of APEC and other agents. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this agent.

亚甲基蓝介导的电化学传感界面PCR鼠伤寒沙门氏菌检测方法

文章研究一种可用于快速检测牛奶中鼠伤寒沙门氏菌(Salmonella typhimurium)的方法,通过在聚合酶链式反应(polymerase chain reaction,PCR)扩增体系中加入嵌入式氧化/还原指示剂亚甲基蓝可以实现对扩增产物的电化学检测。文中所提出的基于丝网印刷电极的电化学检测方法可在120 min内完成对鼠伤寒沙门氏菌的检测,线性范围为3×10^(1)~3×10^(7)CFU/mL,且不受牛奶基质影响。该方法快速、简便、特异性好,有望应用于食源性致病菌现场快速检测。