Development of Species-Specific PCR Primers and Sensitive Detection of the Tylenchulus semipenetrans in China

Tylenchulus semipenetrans is the most economically important and widespread nematode pest of citrus in China.rDNA-ITS of 14 populations of T.semipenetrans which were collected from different citrus groves or Chinese fir（Cunninghamia lanceolata） plantations in China were amplified and sequenced.The species-specific primers were designed for the first time to diagnosis T.semipenetrans based on the sequences of rDNA-ITS regions of geographic population above.The primers were sensitive to amplify the expected band size（297 bp） from DNA template of a single second-stage juvenile（J2） or different life stages of T.semipenetrans.No specific band was amplified from 15 non-target nematode species which were commonly found in citrus groves.Specificity and reliability of the primers were validated by further PCR amplification of 16 extra populations of T.semipenetrans collected from 4 provinces of China.The primers successfully detected a single J2 of T.semipenetrans within a whole nematode community comprising a large numbers of non-target nematode.The developed diagnostic technique can be used for accurate identification of T.semipenetrans and also as a decision tool for nematode management for citrus or Chinese fir in China.

Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer(ITS)region of peritrich ciliates in environmental samples

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifi cations of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specifi c PCR primers were newly designed to amplify a fragment including the internal transcribed spacer(ITS) region of ribosomal rDNA from environmental samples. The primers showed high specifi city in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28 S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18 S rDNA in peritrichs. The newly designed specifi c primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in dif ferent systems.

Species-specific COI primers for rapid identification of a globally significant invasive pest, the cassava mealybug Phenacoccus manihoti Matile-Ferrero

The globally invasive cassava mealybug Phenacoccus manihoti Matile-Ferrero is a pernicious pest of cassava,and its recent introduction into Asia has raised considerable alarm.To slow or prevent further invasion,an accurate,simple,and developmental-stage-independent detection method for P.manihoti is required.In the present study,a PCR method based on a species-specific mitochondrial DNA cytochrome oxidase I(SS-COI)marker was developed for rapid identification of P.manihoti.One pair of SS-COI primers(PMSSZW-1F and PMSSZW-1R)was designed based on sequence variations in the COI gene among P.manihoti and related mealybug species.Specificity of the primer pair was validated on 21 closely related species.Sensitivity tests were performed on four immature developmental stages and female adults.Efficacy tests demonstrated that at the relatively low concentration of(135.2±14.7)pgresuspended DNA,the specific fragment was detected in all replicates.Furthermore,the SS-COI primer pair was assayed on three populations of P.manihoti from major exporting countries of cassava.The PCR assay was proved to be a rapid,simple,and reliable molecular measure for the identification of P.manihoti.This tool will be useful for quarantine,monitoring,and management of this invasive pest.

1B Specific LMW-GS Primers Cloned a 1D Located Gene from Wheat Cultivar Xiaoyan 22

In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.

The application of sequence specific primer and RT-PCR to LRRK2 gene polymorphism typing

Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA markers with known genotypes by use of quantitative fluorescence real-time PCR(RT-PCR).100 cases of PD samples with unknown genotypes were tested,and verified by use of polymerase chain reaction linked restriction fragment length polymorphism(PCR-RLFP).Results:The genotyping results of DNA markers proved to be correct,and 100 cases of samples to be tested had a completely consistent genotyping result with PCR-RLFP genotyping result.Conclusions:Sequence specific primer and quantitative fluorescence RT-PCR can successfully make a genotyping for disease susceptibility loci R1628P and G2385R of LRRK2.

Analysis on 16S-ITS Marker from Bacillus licheniformis and Its Specificity

[Objective] This study aimed to establish molecular identification methods for Bacillus licheniformis. [Method] Based on clone sequencing and difference analysis for 16S and ITS sequences of B. licheniformis TS-01, specific primers were designed using region sequences as the targets used for amplifying all test strains. [Result] The specific primers of B. licheniformis were designed from the ITS and 16S rDNA regions. The optimal annealing temperature of the specific primers for PCR was 67.2 ℃ with 24 cycles. A 905 bp marker fragment was amplified for B. licheniformis TS-01, while all other test strains showed negative results. This indicated that a specific 16S-ITS marker was obtained, which accurately identified the strain at the species level. [Conclusion] This molecular identification method for B. licheniformis TS-01 has laid the foundation for molecular diagnosis of B. licheniformis.

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.

Evolutionary divergence in Chenopodium and validation of SNPs in chloroplast rbcL and matK genes by allele-specific PCR for development of Chenopodium quinoa-specific markers

The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.

广西道地药材肉桂及阴香的DNA分子鉴定研究

建立一种能够准确鉴别肉桂及阴香的特异性聚合酶链式反应方法,以保障肉桂用药的安全性和有效性。通过对比分析肉桂及阴香的psbA-trnH序列差异找到特异性单核苷酸多态性位点,设计特异性引物,通过优化退火温度、循环次数,评估不同聚合酶种类和不同基因扩增仪等扩增条件对不同来源的肉桂及阴香进行特异性扩增,根据特异性扩增条带进行鉴别。结果表明退火温度为54℃,循环次数为35次时,肉桂经肉桂特异性引物扩增后在100~200 bp处出现特异性条带,阴香无条带;退火温度为56℃,循环次数为40次时,阴香经阴香引物扩增后在200~300 bp处出现特异性条带,肉桂无条带。该研究所建立的特异性PCR方法可以快速准确鉴别出肉桂及阴香,为控制肉桂的质量安全提供参考。

基于环境DNA技术的红鳍笛鲷生物量评估方法研究

环境DNA可以定量评估水生生物丰度,但其准确性因引物与构建模式差异而有所不同。以岩礁性鱼类红鳍笛鲷(Lutjanus erythropterus)为对象,采用室内实验和实时荧光定量PCR(qPCR)的方法对红鳍笛鲷引物和探针进行设计与开发,建立DNA浓度与生物量间的关系,解析环境DNA(eDNA)持久性和衰减的过程,初步探究了eDNA技术对红鳍笛鲷生物量评估的可行性。结果显示,当引物退火温度为60℃时,引物扩增效果最好;红鳍笛鲷eDNA浓度与生物量间呈显著正相关(R^(2)=0.93);移出红鳍笛鲷后的20 d内,eDNA衰减浓度与时间呈显著负相关(R^(2)=0.98)。对红鳍笛鲷增殖放流前后的水环境检测结果表明,放流后水体内红鳍笛鲷eDNA浓度显著增加,表明本研究所设计的引物在检测红鳍笛鲷生物量方面的可行性,可为应用eDNA技术开展红鳍笛鲷生物量的准确评估提供理论基础。

采用实时荧光定量PCR法检测大曲中的3种高温放线菌

建立实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)方法对大曲中3种高温放线菌(Thermoactinomyces vulgaris、T.intermedius和T.daqus)进行特异性定量检测。根据T.vulgaris、T.intermedius、T.daqus全基因组中特异性单拷贝核心基因recA、mdH、gyrA为参考序列分别设计了一对可扩增278、233、291 bp片段的引物;经特异性、有效性、准确性实验显示,在常见的13种白酒酿造微生物中,引物TV、TI、TD分别可特异性检测T.vulgaris、T.intermedius、T.daqus,检测范围为2.82~8.82 lg copies/μL,加标回收率95%~105%。进一步对大曲发酵过程样品检测,结果显示,T.vulgaris含量在呈现波动趋势,在第5天达到最高,为(6.06±0.02)lg copies/g;T.intermedius含量呈现先上升后下降的趋势,在第7天达到最高,为(6.33±0.13)lg copies/g;T.daqus含量在0~12 d呈现波动趋势,在12~14 d下降,随后一直上升,发酵结束时含量最高,为(7.62±0.02)lg copies/g。该研究建立的RT-qPCR方法可对大曲发酵过程中3种高温放线菌进行特异性鉴定和快速定量,为进一步监测高温放线菌在白酒酿造过程中的功能提供了方法。

欧李种质资源自交不亲和S-RNase基因的克隆及序列分析

为鉴定不同欧李种质资源的S基因型,分析欧李S基因序列特点,以70份欧李种质为材料,通过PCR特异性扩增S基因,并克隆测序得到其基因序列。结果表明:利用BFP93/BFP94-1引物,在52份种质中各鉴定到2个S基因,在16份种质中各鉴定到单个S基因,在3-32-扁黄和10-32两份种质中未扩增出条带;共克隆得到120条基因,鉴定得到36种S基因型,并将其中的新基因登录到NCBI中,登录号依次为OQ124075~OQ124109。

荧光定量PCR法鉴别蛤蚧定喘丸中醋鳖甲

目的建立蛤蚧定喘丸中醋鳖甲的DNA分子鉴定方法。方法根据鳖、佛罗里达鳖线粒体基因序列的差异设计特异性引物TS和AF,进行荧光定量PCR和方法学考察,然后对市售蛤蚧定喘丸中醋鳖甲进行鉴定。结果含有鳖、佛罗里达鳖的DNA样品仅与相应引物扩增后可检测到荧光信号,有特定温度单一峰,T_(m)分别为77.45、79.72℃。该方法的检测限为10 copies/μL,重复性较高(CV<1%)。10批蛤蚧定喘丸中有9批仅检出鳖DNA,被鉴定为正品;1批同时检出鳖DNA和佛罗里达鳖DNA,被鉴定为掺伪品。结论本研究所建立的方法可快速、可靠地鉴定蛤蚧定喘丸中醋鳖甲炮制品成分,有助于监管含醋鳖甲成方制剂的质量,并为成方制剂中动物药成分的专属性鉴定研究提供新途径。

马乳酒样乳杆菌马乳酒样亚种real-time PCR检测方法的建立与应用

本研究建立了一种特异性实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)检测方法,根据模式菌株马乳酒样乳杆菌马乳酒样亚种ZW3的16S rDNA序列和全基因组序列设计筛选特异性引物,采用SYBR Green I荧光染料建立real-time PCR方法,并对方法的特异性、灵敏度、重复性和混合体系等进行检测。结果表明,本研究所建立的方法特异性强、灵敏度高、重复性好,建立real-time PCR的标准曲线,其决定系数R2为0.965,具有良好的线性关系,且在马乳酒样乳杆菌马乳酒样亚种及混合体系中可以特异性检出。综上,本研究建立的real-time PCR法可以快速、准确地检测马乳酒样乳杆菌马乳酒样亚种,为马乳酒样乳杆菌的特异性定性定量检测提供了一种新的方法。

基于环境DNA的长江中华鲟分布特征探究

中华鲟(Acipenser sinensis)是我国长江流域的旗舰物种,在长江十年禁渔的大背景下,研究中华鲟无损化检测技术具有重要意义。环境DNA(eDNA)技术是一种环境友好型生物监测技术,可以在不直接观察或捕获生物体的情况下对物种进行检测。从文献中筛选出可以用于检测中华鲟eDNA的特异性引物,于2020年9月在长江中下游选取4个中华鲟常出现的区域,进行各断面立体式采样;提取16个点位的e DNA,使用筛选得到的引物对中华鲟进行eDNA的检测,以探究中华鲟的分布特征。结果显示,成功筛选到1组可以检测中华鲟eDNA的引物,使用该引物成功检测到包括中华鲟在内的长江4种鲟类的eDNA,共计测得约300万条鲟类序列。依据测序结果分析不同断面检测到的中华鲟eDNA的差异,发现宜昌江段断面的中华鲟eDNA最多,洞庭湖口断面最少,且表层和底层水体的中华鲟eDNA检出也有显著差异。筛选得到的引物可以用于中华鲟eDNA的检测,中华鲟e DNA的检测结果与中华鲟的历史调查和洄游特征较为吻合。不同水深条件中华鲟eDNA的检出量有显著差异,表明在今后的调查中采用混合或者立体采样可以更加全面地进行中华鲟eDNA的检测。

应用Q-PCR定性检测KIR基因有无方法的建立

目的建立定性检测KIR基因有无的Q-PCR方法。方法根据高分辨水平中国人群KIR等位基因的多态性,并参考国际IPD-KIR数据库,针对16种KIR基因及2DS4-Normal、2DS4-Deleted两种亚型,设计KIR基因特异性引物用于Q-PCR扩增反应;同时设置一孔阴性对照、一孔阳性对照(特异性扩增人体生长激素HGH基因片段),以监控假阳性、假阴性的结果。为验证Q-PCR方法的可靠性,随机选择302份已采用KIR PCR-SSP商品化试剂盒检测的标本,采用Q-PCR方法盲检和对比。结果300人份的Q-PCR检测结果与已知的PCR-SSP检测结果相符,有2份标本结果不一致,其中1例标本的2DS5基因Q-PCR检测结果为阴性,而PCR-SSP检测结果为阳性;另一例标本2DS1基因Q-PCR检测结果为阳性,而PCR-SSP检测结果为阴性。对2份标本分别进行2DS5、2DS1基因测序分型,证实Q-PCR定性检测结果正确。结论本文建立的KIR Q-PCR方法结果准确、可靠,可用于KIR基因有无的定性检测。

杨属派间5个种特异性InDel引物及在派间杂交子代鉴别中的应用

【目的】远缘杂交可以实现有利基因的聚合,是种质创新的重要手段。杨属有些派间杂交困难,且杂交子代从形态学难以区分,本研究开发杨属不同派间的特异性引物,为派间杂交创制新种质的真实性鉴定提供技术支撑。【方法】利用在美洲黑杨和小叶杨中种特异性InDel位点设计引物,并利用SeqHunter2依次比对胡杨、山杨、小叶杨和大叶杨基因组,再用杨属5个派的自然群体材料进行种内保守及种间多态引物的试验验证,筛选出杨属不同派的特异性引物序列,对杨属不同派间杂交子代进行真实性鉴别。【结果】将从美洲黑杨基因组设计的5000对InDel引物依次分别比对胡杨、山杨、小叶杨和大叶杨基因组,最后筛选出341对引物在上述5个种中是通用的。从不同染色体上共选取48对引物并合成,利用5个树种各1个DNA检测得到43对引物是通用的,其中19对引物在不同种中扩增条带单一且存在差异。利用自然群体混合DNA对试验验证的19对通用引物进行种间多态性和种内保守性检测,初步获得4对多态性引物。最后再用自然群体DNA对这4对引物进行扩增检测,发现引物Chr12_4341229和Chr13_10582756在美洲黑杨、小叶杨、山杨、大叶杨和胡杨中均只能扩增出一个位点,引物Chr12_4341229扩增条带分别为148、152、113、152和159 bp,引物Chr13_10582756扩增条带分别为140、144、116、140和159 bp。利用上述2对引物对小胡杨2号、黑胡杨和黑小杨进行鉴定,发现所有杂交子代个体均能检测它们2个亲本的特征带。引物Chr12_4341229和Chr13_10582756一起使用,可以用于美洲黑杨、小叶杨、山杨、大叶杨和胡杨任意组合杂交子代的真实性鉴别。【结论】本研究获得了杨属5个派间的5个种的特异性InDel引物2对,可以有效地进行杨属不同派5个种之间任意组合种间杂交子代的真实性鉴别,为进一步开展杨树派间杂交创制新种质提供了技术支持。

鸡圆圈病毒3型可视化LAMP检测方法的建立及应用

建立鸡圆圈病毒3型(GyV3)可视化环介导等温扩增(LAMP)检测方法,为临床上快速检测鸡GyV3提供一种诊断方法。参考GyV3的VP3基因序列,设计1套LAMP特异性引物,分别通过温度优化、引物浓度优化和反应时间的优化,确定鸡GyV3可视化LAMP诊断方法的扩增温度、引物浓度和反应时间,通过特异性试验、敏感性试验、重复性试验和临床样品检测,验证鸡GyV3可视化LAMP诊断方法的特异性、敏感性、稳定性和可靠性。结果显示,鸡GyV3 LAMP检测方法的反应体系:总体积为20.0μL,包括DNA模板1.0μL,2×Master Mix 10.0μL,内引物GyV3-FIP和GyV3-BIP混合液(工作浓度16.0μmol/L)2.0μL,外引物GyV3-F3和GyV3-B3混合液(工作浓度2.0μmol/L)2.0μL,环引物GyV3-LF和GyV3-LB混合液(工作浓度8.0μmol/L)2.0μL,ddH_(2)O 3μL;鸡GyV3 LAMP检测方法的扩增程序:66℃反应20 min,80℃灭活5 min。鸡GyV3可视化LAMP检测方法能特异性检测GyV3,最低检测浓度为10^(1)拷贝/μL,组内和组间变异系数小于5%;与常规PCR方法检测临床样品的结果比较,两者结果符合率达100%。建立的鸡GyV3可视化LAMP检测方法能特异性检测GyV3,具有特异性好、敏感性高及污染小等优点,且检测结果可通过肉眼观察进行判断,适用于GyV3的临床快速筛查。

簸箕柳×三蕊柳种特异性KASP标记开发及种间杂交子代鉴别

【目的】开发簸箕柳和三蕊柳种特异性KASP引物,为开展簸箕柳和三蕊柳杂交选育速生、抗虫新种质的真实性鉴定提供参考。【方法】利用簸箕柳和三蕊柳重测序数据,开展全基因组SNP位点分析,筛选出种特异性的SNP位点并设计KASP引物,利用SeqHunter2检测设计引物在簸箕柳中的通用性,并利用簸箕柳和三蕊柳自然群体材料对合成的引物进行实验验证,筛选出簸箕柳和三蕊柳种特异性KASP引物,并对它们的杂交子代进行真实性鉴别。【结果】将三蕊柳和簸箕柳重测序的数据比对到三蕊柳基因组,共获得个6598144个SNPs。经过筛选,最终在簸箕柳中检测到674144个和三蕊柳相比为纯合突变的位点。从每条染色体上随机选取100个位点用于KASP引物设计,在1900个SNPs位点中,750个SNP位点可以成功设计出KASP引物。从每条染色体上选取2组,共选取38组引物,通过SeqHunter2检测得到11组引物在簸箕柳中是通用的。利用簸箕柳和三蕊柳自然群体DNA对合成的10组通用引物进行种内保守和种间差异性检测,获得4组在簸箕柳、三蕊柳和种间杂交子代中聚类明显的引物。引物Stri08_82809、Stri14_11602、Stri14_12274和Stri17_10731在簸箕柳自然群体中分别扩增出纯合G/G、T/T、A/A和T/T位点,在三蕊柳自然群体分别扩增出纯合A/A、C/C、G/G和C/C位点,而在真实杂交子代中分别扩增出G/A、T/C、A/G和T/C杂合位点。利用上述4组引物对簸箕柳×三蕊柳的杂交子代进行鉴定,发现在31个杂交子代中,29个个体扩增出杂合位点,说明同时遗传了2个亲本的特征位点,为真实的种间杂交子代。有2个个体扩增的位点为纯合且和母本相同,说明这2个子代为簸箕柳花粉污染的子代。【结论】本研究获得了簸箕柳和三蕊柳种间特异性KASP标记4组,可以准确快速的开展簸箕柳和三蕊柳种间杂交子代的真实性鉴别,为进一步簸箕柳和三蕊柳种间杂交及回交创制抗虫新品种提供了技术支持。

基于酶介导双重指数扩增技术(EmDEA)的小火蚁快速检测方法的开发

小火蚁Wasmannia auropunctata是危害性最严重的入侵蚂蚁之一,对动植物、生态、经济甚至是人类都容易造成严重影响,是我国的重大检疫害虫之一。本研究为实现小火蚁的现场快速精准检测,通过自研程序筛选出小火蚁全基因组中的特异性片段,具有更高的特异性,基于酶介导双重指数扩增技术(EmDEA)筛选了适用的引物、RNA探针,全部反应所需时间小于20 min,并验证了其特异性和灵敏度。结果显示这种方法可在短时间内达到极高检测灵敏度与特异性,最低检出限约为4 ng。这种方法极大程度降低对设备的依赖,简单、快速、特异,为小火蚁的基层检测与现场诊断提供技术支持。