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.

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.

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.

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.

A Rapid Identification Method of Bactrocera cilifera (Hendel) with Species-Specific Primers(SS-COI)

[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit flies such as B.diaphora(Coquillett)and B.dorsalis(Hendel)as the negative controls,a pair of species-specific primers,YF290 and YR511,were designed and screened for accurate identification of B.cilifera,based on mitochondrial DNA COI sequence.[Result]The PCR products were amplified and detected by electrophoresis.Only a clear and single band was observed at about 222 bp in the positive control,while no bands were found in the other negative controls.[Conclusion]The established rapid identification method with species-specific primers(SS-COI)is of great practical significance for rapid identification of fruit flies intercepted from import and export fruits and vegetables at ports,and for rapid clearance and early warning of import fruits and vegetables at ports.

Primers for the Amplification of the Circular Chloroplast DNA from the A-genome Group of Cultivated Cotton

The availability of the plastid genome sequences is one of the bases for comparative,functional,and structural genomic studies of plastid-containing living organisms,in addition to the application

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.

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.

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.

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.

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.

Molecular Marker Techniques Using Single Primers and Their Advances

Molecular marker techniques have been widely applied in the fields of genetic diversity analysis,germplasm resources identification,molecular fingerprint and genetic linkage map construction,QTL mapping and molecular assisted breeding.On the basis of stating the concept of molecular marker techniques based on single primer amplification reactions,this study focused on the sorting and induction of single-primer molecular marker techniques,and expounded their derivative development.Finally,the application prospect and future expectation of single-primer molecular marker techniques were described in detail.The purpose of this study was to clarify the types of molecular marker techniques based on single primer amplification reactions,so that researchers can quickly and conveniently select molecular marker techniques according to their own specific scientific research conditions.

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.

Using homology primers to clone a novel gene of β-chemokine receptorsupergene-family

Objective Using homology primers for clone of β-chemokine receptor supergene-family from lower level RNA expression. Methods: Through similarity alignment of the conservative transmembrane domainof human MCP-l receptor, mouse MIP-lα receptor and rat IL-8 receptor. the degenerate oligonucleotide homology primers were designed and a cDNA fragment was amplified by RT--PCR. After a new gene sequenceof chemokine receptor supergene-family in GenBank was identified, the new gene full sequence was cloned bya Marathon PCR of the specific primers. Results: Mouse CCR5 full-length cDNA with 2888 bp was cloned,including open reading frame 1065 oligonucleotides and coding 355 amino acids. This gene was a novel member of the β-chemokine receptor supergene-family confirmed by sequence analysis, ligand binding assay andinforming from GenBank. Conclusion: Using homology primers, the novel member of the supergene-familycan be captured. This method is a new cloning strategy and has extensive significance in the molecular cloningof a new gene.