应用PCR及Nested-PCR技术检测柑橘黄龙病病原研究

以采自田间和广东省农业科学院果树研究所防虫隔离网室内嫁接并感染黄龙病的5个柑橘品种叶片为材料,以草本指示植物长春花(Catharanthusroseas)和木本指示植物柑(CitrusreticulataBlanco)鉴定为基础,采用改良的CTAB法提取总DNA,在此基础上进行常规PCR与NestedPCR检测,并对特异目的片段进行克隆、测序。试验证明NestedPCR比常规PCR的灵敏度更高。这两种方法均可以检测出未显症的黄龙病材料,但NestedPCR可以在木本指示植物嫁接后40d左右、草本指示植物摩擦接种1个月左右检测到病原;而常规PCR在木本植物嫁接后60d左右、草本植物摩擦接种近2个月左右才能检测到病原。常规PCR所能检测到的最低DNA的量为pg数量级;NestedPCR检测病原DNA灵敏度约为常规PCR的104倍。

广西砂糖橘黄龙病亚洲种的Nested-PCR检测

【目的】利用柑橘黄龙病病原亚洲种16S rDNA序列的特异引物检测疑似黄龙病的砂糖橘叶片样品,为砂糖橘黄龙病的综合防控提供理论依据。【方法】选取采自广西9市17个采样点的214份疑似黄龙病及无症状砂糖橘叶片样品,用试剂盒法提取叶脉基因组DNA,采用柑橘黄龙病亚洲种16S rDNA特异引物进行PCR扩增,扩增结果在1%琼脂糖凝胶电泳上检测,统计待检样品柑橘黄龙病病原菌亚洲种的带菌率。【结果】对214份样品的Nested-PCR扩增结果,部分样品可得到1160 bp左右的特异条带,与预期大小相符但条带明暗不同。来自广西9市17个采样点的砂糖橘叶片样品均检测出黄龙病亚洲种病原,平均检出率均在66.0%以上,其中检出率最高的是来自东兴市的样品,检出率为100.0%,检出率最低的是来自南宁市的样品,检出率为66.9%;斑驳、缺素型黄化、叶脉黄化、小叶和无症状叶片样品中均可检测出黄龙病亚洲种。【结论】Nested-PCR技术具有灵敏度高、特异性强的特点,能够快速、准确检测出各种症状样品的黄龙病病原,可用于柑橘黄龙病的早期诊断。

禽弱毒疫苗中鸡传染性贫血病病毒nested-PCR检测方法的建立

疫苗中污染鸡传染性贫血病毒(CIAV)是造成CIAV感染的途径之一,而通常疫苗中CIAV污染剂量较低,应用一般的技术难以检测到。为了更灵敏地检测禽弱毒疫苗中CIAV的污染,根据Gen Bank已发表的CIAV基因序列,针对其保守区设计了外用引物和内用引物,通过优化反应体系和反应条件建立了检测CIAV的nested-PCR检测方法。结果显示,所建立的检测方法灵敏度比常规PCR高100倍,该方法可以检测到1 000羽份弱毒疫苗中1 EID_(50)的低剂量CIAV污染,应用该方法从送检的50个疫苗样品中检测出4个样品为CIAV阳性。该方法与禽白血病病毒、禽网状内皮组织增生症病毒以及马立克氏病病毒等的基因均无交叉反应,具有很好的特异性。提示所建立的nested-PCR灵敏度高、特异性强,可用于检测禽弱毒疫苗中CIAV低剂量的污染。

Development and Clinical Application of a Single-tube Nested PCR Method to Amplify the DNA Polymerase Ⅰ Gene of Treponema Pallidum

Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.

One tube nested-PCR在SNP基因型分型中的应用

nested–PCR是一种在普通PCR基础上发展起来的专门用于检测单核苷酸多态性(SNP)的技术。结合nested–PCR技术在水稻中检测SNP的研究,以1个水稻香味基因Fgr和3个稻瘟病基因Pi–ta、Pi9、Pigm为例,把nested–PCR的4条引物同时加在一管PCR反应中进行扩增,在其序列内针对SNP位点设计功能标记,用来扩增含有突变位点的DNA片段。通过优化引物浓度梯度和改良反应程序,达到了一步快速检测SNP基因型的目的。

Development and Application of Nested PCR Assay for Detection of Dairy Cattle-Derived Cyclospora sp.

To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp., two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp. sequences. These primers selectively amplified a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. With these primers, a nested PCR assay for the detection of cattlederived Cyclospora sp. was developed. The nested PCR assay was specific and there is no cross-reaction with other parasites, such as Eimeria spp., Cryptosporidium spp., Giardia sp., Toxoplasma sp., Trichuris sp. and cattle ciliate. The assay was able to detect as low as 2.85 × 10^-2 fg of the control positive DNA. The results of the detection of clinical samples indicated that the assay coincided with microscopic examination. The results show that the nested PCR assay will be an effective tool for the detection of Cyclospora sp. in cattle feces.

Nested multiplex PCR for identification and detection of human Plasmodium species including Plasmodium knowlesi

Objective:To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria.Methods:In this study the nested multiplex malaria PCR was redesigned,targeting the 18S rR NA gene,to identify the fifth human Plasmodium species,Plasmodium knowlesi,together with the other human Plasmodium(Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale and Plasmodium malariae)by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives.Results:The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms.The technique showed high sensitivity(100%)and specificity(96%)when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos栿and a published real-time PCR malaria assay.Conclusions:The technique designed is an economical,sensitive and specific alternative to current diagnosis methods.Furthermore,the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.

泰泽病原体nested-PCR检测方法的优化及应用研究

目的对泰泽病原体nested—PCR检测方法进行优化和应用研究。方法以感染泰泽病原体RJ株的大鼠肝组织作为阳性对照，按国标GB/T 14926．10—2008《实验动物泰泽病原体检测方法》间接免疫荧光检测fIFA）呈阳性的上海地区大鼠肝组织为材料，对本室已建立的泰泽病原体nested—PCR检测方法中的DNA抽提方法和PCR过程进行优化，并应用于22只免疫抑制实验动物的泰泽病原体检测。结果肝脏组织DNA以酚／氯仿抽提法为好；nested—PCR出现196bp的特异性条带，此方法的敏感性达1pg；产物纯化后进行测序比对分析显示，上海地区实验大鼠感染样品以及RJ株感染样品的序列均与Genbank中泰泽病原体16S rRNA（Genbank D14638．1）的同源性为100％。应用研究表明，22个样品中IFA检测阳性样品8个，阳性率为36．4％；Nested-PCR检测阳性样品13个，包含所有的IFA阳性样品，阳性率为59．1％。结论本方法灵敏度高，特异性强，经进一步应用改进后，可用于泰泽病原体的检测。

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.

Development and Application of the Nested PCR Assay for Duck Flavivirus

[ Objective] Aim to establish a kind of efficient detection method for duck flavivirus（DFV）. E Method] The method of nested PCR based on flavivirus universal primers which were designed according to the GeneBank flavivirus gene sequence. [ Result] The degenerate universal prim ers Flav P1 -Flav P4 were designed by genome comparison to target NS5 gene conserved area. The system could only amplify flavivirus purpose gene and the sensitivity was 90 copies/iJL, higher than the ordinary PCR 1 000 times. Homology and evolutionary analysis showed that duck flavivirus belonged to mosquito-born flavivirus, NTAV group, similar with Tembusu and BYD virus. [ Conclusion] Primers of the nested PCR system had good universality and specificity and method had high sensitivity. This system successfully detected flavivirus and clarified the evolution station of DFV.

黄檗丛枝菌根(AM)真菌PCR-DGGE条件研究

从东北林业大学林场采集黄檗根际土壤及菌根，采用Nested—PCR技术分别扩增黄檗菌根及根际土壤AM真菌18SrDNANS31／Glol区域，利用该扩增产物对AM真菌DGGE条件进行优化。表明Nested—PCR技术具有较高的灵敏性，可有效的从微量DNA中扩增出约230bp的目标片段；经DGGE条件优化，确定AM真菌最佳DGGE条件为丙烯酰胺浓度8．0％，纯化后的PCR产物作为DGGE上样样品，凝胶变性梯度范围30％～60％，电泳电压130V，电泳时间8h，电泳温度60℃。

蜜蜂囊状幼虫病毒病的Nest-PCR检测

蜜蜂的囊状幼虫病是中华蜜蜂常见且危害严重的疾病,病原为一小正链RNA病毒。如何尽早鉴别蜂群的感染在养蜂业生产上具有重要的意义。本文报道一种利用RNA反转录结合巢式PCR检测鉴定蜜蜂的囊状幼虫病毒病的方法。

血清登革热病毒NEST-PCR检测

目的 建立用NEST—PCR快速检测登革热病毒的方法。方法 通用引物对样本进行RT—PCR扩增，然后用分型引物进行NEST—PCR，根据产物条带的位置分型。结果 标准毒株的扩增结果与预期结果相同，血清登革热病毒在病程2～5d的阳性率高于6～9d的阳性率，分别为6／7和2／6。结论 NEST—PCR方法是一个快速、准确检测登革热的方法，但它更适用于早期临床检测。

套式实时荧光定量PCR检测人外周血单个核细胞中FOXP3 mRNA

目的建立检测人外周血单个核细胞(PBMC)中FOXP3mRNA的方法。方法跨内含子设计外引物,并根据扩增的片段,设计一对内引物和TaqM an探针,采用套式实时荧光定量PCR(nested-real-tim e-PCR)方法。首次PCR扩增用减少引物浓度及循环次数的方法以增加其模板的特异性,再行第二次PCR扩增作实时荧光定量检测,用FOXP3和β-actin标准品作标准曲线,以二者比值作为FOXP3mRNA表达水平指标。结果重复6次PCR,6次试验C t的总平均值为14.66,平均变异系数为5.81%。C t值和模板起始浓度对数值之间具有较好的线性关系(R2=0.999);检测最低浓度可达100拷贝。结论nested-re-al-tim e-PCR检测PBMC中FOXP3mRNA的方法简单且重复性好,高度敏感且特异,可作为评价免疫功能及某些疾病治疗效果观察的临床常用方法。

Detection of Adenovirus in Fresh Fruit, Vegetables, Wastewater and Manure from Irrigated Farms in Ouagadougou, Burkina Faso

Enteric viral pathogens are responsible for numerous epidemics associated with the consumption of fresh fruit and vegetable, whether raw or minimally processed. The aim of the present study was to assess agricultural practices and the presence of adenovirus (AdV) in fruits and vegetables, manure and irrigation wastewater sampled in the urban and peri-urban perimeters of Ouagadougou. A total of 286 samples including 30 lettuces, 42 tomatoes, 30 carrots, 30 strawberries, 74 manures and 80 wastewater samples were collected from four market garden sites in and around Ouagadougou. Nested PCR was performed with specific primers to detect adenoviruses (AdVs). A face-to-face survey was carried out using a questionnaire on market garden production practices. Overall, adenoviruses prevalence was 5.9% [IC95, 3.2% - 8.7%] in all samples analyzed. It was specifically 7.14% (3/42) from tomatoes, 6.7% (2/30) from lettuces, 20% (6/30) on strawberries and 7.5% (6/80) in irrigation water. The survey showed that irrigation water came from untreated sources (dam, well, canal) and then 52% of farms used untreated manure. No farms have implemented measures to limit access by domestic and wild animals. This work shows the presence of human adenoviruses in surface irrigation water and fresh produce, which is of concern when fresh produce is consumed raw. To reduce the public health risks associated with consuming these foods, it is essential to follow good hygiene and cultivation practices.

湛江农垦红江橙主产区2个果园黄龙病的巢式PCR检测分析

柑橘黄龙病(HLB)是柑橘生产上的一种毁灭性病害,对柑橘生产为害较重。为了明确柑橘黄龙病在红江橙果园中的分布及危害情况,进一步为柑橘黄龙病的综合防控提供有价值的参考,采用Nested PCR分子检测技术,于2016~2018年对湛江农垦红江橙主产区具有黄化、斑驳等症状的305个红江橙样本进行检测。结果表明,305个红江橙叶片样本中,HLB的阳性检出率为53.1%;其中红江农场38队红江橙果园黄龙病的阳性检出率为55.6%,广垦(湛江)红江橙农业科技有限公司横江红江橙果园黄龙病的阳性检出率为48.6%。柑橘黄龙病已在湛江农垦红江橙果园普遍发生,生产上应引起重视。

Clinical Identification of Common Species of Dermatophytes by PCR and PCR-RFLP

Summary: To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting TopoisomeraseⅡgene. The DNA of 7 dermatophytes, along with Candida albicans, Aspergillus terreus and Aspergillus flavus were amplified by consensus primer dPsD1. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme HincⅡ. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect for T. mentagrophytes and T. tonsurans. From the restriction profiles of HincⅡ, 6 of the 7 dermatophytoses were diagnosed to species level including T. mentagrophytes and T. tonsurans. By combining the results of the PCR and PCR-RFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase Ⅱ gene is rapid and efficient.

A Study on Diagnosis of Mycoplasma Hominis in The Urogenital Tract By Nested Polymerase Chain Reaction with First Void Urine in Men

Objectives: To evaluate thc efficacy of nested polymerasechain reaction (PCR) with first void urine (FVU) for thediagnosis of Mycoplasma hominis in male patients. Methods: Matched FVU specimens and urethral swabs werecollected from 194 male patients with NongonococcalUrethritis and tested by nested PCR and cell culture. Cellculture was used as a gold standard for evaluating other assaytechniques. Results: For FVU nested PCR assay and FVU cell culture,our results showed that the sensitivity was 100% and 93.3%;specificity was 97.0% and 98.2%; positive predictive value(PPV) was 85.7% and 90.3%, negative predictive value (NPV)was 100% and 98.8%, respectively. The total consistencybetween the two techniques was 97.4%. Conclusions: For the diagnosis of Mycoplasma hominis inmen, nested PCR detecting FVU is a highly sensitive andspecific method. First void urine can replace swab culture orPCR in terms of acceptability and feasibility.

传染性法氏囊病快速诊断技术的建立及其应用

根据已发表的IBDV各致病型毒株的序列,在病毒结构蛋白VP2编码基因的高变区(VP2variabledo-main,vVP2)两端外侧的保守序列内设计合成了2条寡核苷酸引物,对各种不同致病型的12株参考毒株进行逆转录酶—聚合酶链式反应(RT-PCR)的检测.结果12个参考毒株均能扩增出约679bp的目的片段,而对作为阴性对照的常见5种鸡病病原NDV、IBV、CAIV、E.coli、pM均没有扩增出任何片段;应用建立的技术对疑似IBD的34份临床病料进行检测,并同时在基础RT-PCR扩增的片段内设计另一对引物进行Nested-PCR检测.结果基础RT-PCR方法检测到11份阳性,Nested-PCR检测到23份阳性.研究的结果表明建立的IBD的RT-PCR诊断技术具有特异、快捷、敏感的特点.在此基础上设计的Nested-PCR则大大提高了阳性检出率(提高52.1%).

黄檗丛枝菌根真菌鉴定

目的:利用形态学特征与Nested-PCR技术鉴定黄檗丛枝菌根真菌。方法:采用酸性品红染色法挑选黄檗丛枝菌根。同时,利用湿筛法获得AM真菌孢子,进行形态学鉴定。运用Nested-PCR技术,对黄檗粗提DNA进行特异性扩增,采用blastn进行序列相似性比较,并构建系统进化树,确定侵染黄檗根系的AM真菌。结果:编号为HDAM-1的AM真菌孢子,形态特征与G.intraradices的特征描述一致。Nested-PCR检测到约455bp的目的片段,其序列与G.intraradices(DQ469118)相似性最高,达97.8%,有11个碱基的差异。系统进化树显示该序列在基于25SrDNA的进化树中与G.intraradices(DQ469118.1)处于同一分支,确定G.intraradices侵染黄檗根系。结论:将形态学特征与Nested-PCR技术相结合鉴定AM真菌,不仅简易、经济,而且能够提高研究结果的可靠性。