3'-terminus shifted bases degeneracy primers increasing sensitivity of polymerase chain reaction

目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。

Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length sequences.Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs.The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 phyla.RESULTS:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 nt.Among 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on classification.In comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 sequences.The 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species level.Weighted by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species.Assuming that a typicalpolymerase chain reaction can tolerate 2 mismatches between a primer and a template,the modified 347F and 803R primers should be able to anneal 98%and 99.6%of all 16S rRNA genes in the RDP database.CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.

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.

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.

Studies on the Primers Screening for AFLP Fingerprints of Rice Cultivars

AFLP(amplified fragment length polymorphism) is a very powerful fingerprinting technology. The key of making variety fingerprints is to select specific powerful primers for each crop. A quick and effective procedure for selecting AFLP primers for rice variety fingerprinting was established as the following: (1) Choose3 or more group materials that have close genetic relations. (2) Select potential polymorphic primers from primer pairs that are 2 + 2 primer crosses and same at two ends. (3) Recombine the selected potential polymorphic primers and choosing more polymorphic primers. (4) Add one selecting base at one end to become 2 + 3 or 3 + 2 primers and further selecting more polymorphic primers. Some primers were selected with this procedure, such as M21 P87 and M73P17, with which the fingerprints had more polymorphism and high quality.

An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens

Objective To provide a feasible and cost-effective next-generation sequencing （NGS） method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. Methods Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure： reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide （random hexamer or non-ribosomal hexanucleotide） primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. Results NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease （HFMD） and identified other potential viruses or differentiated misdiagnosis events. Conclusion The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.

Design of Vibrio 16S rRNA Gene Specific Primers and Their Application in the Analysis of Seawater Vibrio Community

类 Vibrio 原因 vibriosis 的病原的种， maricultured 动物和海味消费者的最流行的疾病之一。在海味生产的链监视他们的动力学，处理并且消费为食物和海洋生物养殖安全是很重要的。为了充实代表 Vibrio 16S ribosomal RNA 基因(rDNA ) ，碎裂并且识别进一步真实及时的这些细菌并且同时地在在链的细菌的植物群之中，有选择地放大 Vibrio 16S rDNA 碎片的一双教材与他们在海水 Vibrio 的分析证明的特性和报道被设计社区。二份教材， VF169 和 VR744 的 Thespecificities 和范围，被使用强风节目在在 GenBank 可得到的细菌的 16S rDNAs 之中理论上决定，实际上，由放大 Vibrio16S， rDNA 从海水 DNA 碎裂。超过 88.3% 序列在 GenBank，与 VR744 显示出相同火柴，属于 Vibrio 类。33 克隆的一个总数随机被选择并且定序。所有序列显示出他们的最高的类似到并且多样的已知的 Vibrio 种类在那些附近聚类。设计的教材能够检索宽范围 ofVibrio 16S rDNA 在海水在细菌的植物群之中明确地碎裂，海味耕作的最重要的自然环境。

Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment

AIM: To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV to tyrosine-valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-aspartate-aspartate (YIDD).METHODS: Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration, and annealing temperature of primers were systematically examined. Pair primers specifi c to rtL180M and rtM204V were selected for YVDD detection. Primer specif ic to rtM204I with an additional 3’-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment.RESULTS: We could detect as little as 0.001%-0.00001% of mutant viruses coexisting in 108-109 copies of wild-type HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAg-negative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations. CONCLUSION: We developed a highly sensitive and specifi c assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment.

Preliminary Study on Applicability of Microsatellite DNA Primers from Parasite Protozoa Trypanosoma cruzi in Free-living Protozoa

In this paper, we took the lead in studying on specificity of the microsatellite DNA loci and applicability of mi crosatellite DNA primers in protozoa. In order to study characters of microsatellites in free living protozoa, eight microsatellite loci primers developed from Trypanosoma cruzi (MCLE01, SCLE10, MCLE08, SCLE11, MCLF10, MCLG10, MCL03, MCL05) were employed to amplify microsatellite in four free living protozoa, including Bodo designis, Euglena gracilis FACHB848, Paramecium bruzise and Tetrahymena thermophila BF1. In the amplification systems of P. bruzise, four loci (SCLE10, SCLE11, MCLF10, MCL03) were amplified successfully, and four amplification fragments were in proper size. In genome of E. gracilis FACHB848, five of eight primers brought five clear amplification bands. In B. designis, three (No.4, 5 and 7) of eight loci produced clear and sharp products without stutter bands, whereas no bands appeared in T. thermophila BF1. Further, eight 300-500 bp amplification fragments were cloned and sequenced. Nevertheless, all sequenced products did not contain corresponding microsatellite sequence, although Bodo is in the same order and has the nearest phylogenetic relation with Trypanosoma among these four species. Thus, the microsatellite DNA primers can not be applied among order or more far taxa, and the specificity of microsatellite DNA is very high in protozoa. The results of this study will contribute to our understanding of microsatellite DNA in protozoa.

Heavy metal free primers: Polymorphism of gadolinium and titanium in the context of GSR glass phase

The possibility of identifying gunshot residue (GSR) particles produced by non-toxic primers containing only titanium and zinc is a very difficult task using SEM/EDX analysis employed in the analysis of GSR originating from primers containing lead, barium and antimony. However, Bauer et al. demonstrated that non-toxic (TieZn) primers form a TiZn2O4 spinel crystalline structure using SEM/EDX with EBSD (Electron Back Scatter Diffraction) and TKD (Transmission Kikuchi Diffraction), whereas GSR originating from gadolinium-doped TieZn primers form a non-crystalline glass phase. Here, a possible explanation of these different phenomena is hypothesized.

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing was developed by a synthesis method （DP-SBS） for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number （n） of degenerate nucleotides at the 3＇-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides （n＋1） on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.

Specific primers design based on the superoxide dismutase b gene for Trypanosoma cruzi as a screening tool:Validation method using strains from Colombia classified according to their discrete typing unit

Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biological samples.Methods:Strains were isolated,DNA extracted,and classified by using three Polymerase Chain Reactions(PCR).Subsequently this DNA was used along with other isolates of various biological samples,for a new PCR using primers designed.Finally,the amplified fragments were sequenced.Results:It was observed the predominance of DTU i in Colombia,as well as the specificity of our primers for detection of T.cruzi,while no band was obtained when other species were used.Conclusions:This work reveals the genetic variability of 21 new isolates of T.cruzi in Colombia.Our primers confirmed their specificity for detecting the presence of T.cruzi.

Comparison of Extraction Methods for Genomic DNA of Fall Webworm [Hyphantria cunea( Drury) ] and Amplification Effect of Different Primers on Sequences of CO I and CO II and Cytb

[ Objective ] The paper was to compare different extraction methods for genomie DNA of fall webworm [ Hyphantria cunea （Drury） ] and study the am- plification effect of different primers on sequences of CO I and CO II and Cytb. [Method] With winter pupae of fall webworm as the test materials, genomic DNA of fall webworm were extracted by five different methods including CTAB method, SDS method, improved SDS method, kit method and different processing time. Six groups of different primers were used to amplify sequences of CO I and CO II and Cytb. [ Result~ Full DNA could be extracted by CTAB method, SDS method and improved SDS method, and the effort of low concentration SDS method was the best. Six groups of primers could amplify the corresponding fragment, and two groups of primers had the best effort for amplification of CO I . [ Conclusion] The study provides better methods for DNA extraction.

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.

Screening of Primers for DNA Barcoding of Sedum Plants

[ Objective] This study aimed to screen primers for DNA barcoding of Sedum plants. [ Method] Sedum lineare Thunb., S. spectabile Boreau, S. ta- tarinowii Maxim, S. aizoon L. and S. sarmentosum Bunge were used as experimental materials. Nuclear DNA and chloroplast DNA were extracted from these five Sedum plants with CTAB method for primer screening by PCR amplification. PCR products were detected by agarose gel electrophoresis. [ Result] Four sequences suitable for DNA barcoding of Sedum plants were preliminarily screened from six candidate sequences （psbA-trnH, ropB, rbcL, rpoC1, ITS 2, marK）, including psbA-trnH, ropB, rpoC1 and ITS 2. The amplification rate of these four sequences reached 100%. [ Conclusion] This study provided basis for DNA barcoding of Sedum plants.

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.

Screening of SSR Core Primers and Construction of Molecular ID for Flue-cured Tobacco Germplasm Resources in Hunan

[Objectives]This study was conducted to establish a reliable and unique molecular ID for flue-cured tobacco germplasm resources in Hunan Province,further improving the efficiency of germplasm collection and identification,and laying a solid material foundation for flue-cured tobacco breeding.[Methods]Twelve pairs of SSR primers with stable amplification and rich polymorphism were screened out from 816 pairs of SSR primers by a step-by-step screening method.As core primers of the SSR core primer library,the polymorphism of SSR primers was analyzed,the genetic relationship of 162 flue-cured tobacco germplasm resources was identified,and the molecular ID cards were constructed.[Results]The result of SSR primer polymorphism analysis showed that a total of 57 alleles were detected by 12 pairs of SSR primers in 165 tobacco germplasm resources,with an average of 4.75 alleles per pair of primers;the average diversity of SSR primers was 0.649;and the average value of Shannon's index was 1.235.The results of cluster analysis showed that 162 flue-cured tobacco germplasm resources were divided into five groups.The members of each group were divided based on genome information,which had nothing to do with their geographical origin.Meanwhile,12 pairs of SSR primers gave each flue-cured tobacco germplasm resource a unique molecular ID code.[Conclusions]From the above results,we can see that the 12 pairs of SSR primers obtained by screening have stable amplification polymorphism,and can serve as the primers of the core primer library,and can be used to construct the unique molecular ID of flue-cured tobacco germplasm resources.

Effect of Universal Primers on the Tensile Bond Strength between Zirconia and Resin Composites

This study aimed to evaluate the effects of universal primers on the tensile bond strength between zirconia and resin composites.Zirconia specimens were divided into five groups based on the surface treatment with the following primers:MP(Monobond Plus),SU(ScotchBond Universal),AZ(AZ Primer),BM(Beauty bond Multi),and BL(Bondmer Lightless).After priming,stainless steel rods were bonded to the zirconia specimens with composite resin.The tensile bond strength test was performed:stored at room temperature for 1 day;stored in distilled water at 37°C for 7 days;and underwent thermal cycling.The BL group demonstrated a significantly higher tensile bond strength than other groups when stored at room temperature for 1 day(p<0.05).The primer that acted via chemical polymerization appeared to be most effective in improving the bond strength between the two materials in this study.