scholarly journals Specific DNA identification of Pheretima in the Naoxintong capsule

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Xiaoxiao Zhu ◽  
Hoi-Yan Wu ◽  
Pang-Chui Shaw ◽  
Wei Peng ◽  
Weiwei Su
Keyword(s):  
Pcr Amplification ◽  
Cerebrovascular Diseases ◽  
Coi Gene ◽  
Chemical Marker ◽  
Dna Identification ◽  
Specific Primers ◽  
Blast Search ◽  
Pcr Products ◽  
Crude Drugs ◽  
Nucleotide Database

Abstract Background Pheretima is a minister drug in Naoxintong capsule (NXTC), a well-known traditional Chinese medicine (TCM) formula for the treatment of cardiovascular and cerebrovascular diseases. Owing to the loss of morphological and microscopic characteristics and the lack of recognized chemical marker, it is difficult to identify Pheretima in NXTC. This study aims to evaluate the feasibility of using DNA techniques to authenticate Pheretima, especially when it is processed into NXTC. Methods DNA was extracted from crude drugs of the genuine and adulterant species, as well as nine batches of NXTCs. Based on mitochondrial cytochrome c oxidase subunit I (COI) gene, specific primers were designed for two genera of genuine species, Metaphire and Amynthas, respectively. PCR amplification was performed with the designed primers on crude drugs of Pheretima and NXTCs. The purified PCR products were sequenced and the obtained sequences were identified to species level with top hit of similarity with BLAST against GenBank nucleotide database. Results Primers MF2R2 and AF3R1 could amplify specific DNA fragments with sizes around 230–250 bp, both in crude drugs and NXTC. With sequencing and the BLAST search, identities of the tested samples were found. Conclusion This study indicated that the molecular approach is effective for identifying Pheretima in NXTC. Therefore, DNA identification may contribute to the quality control and assurance of NXTC.

scholarly journals First Report of Anthracnose Caused by Glomerella acutata on Chili Pepper in China

Plant Disease ◽  
2011 ◽  
Vol 95 (2) ◽  
pp. 219-219 ◽  
Author(s):  
H. Xia ◽  
X.-L. Wang ◽  
H.-J. Zhu ◽  
B.-D. Gao
Keyword(s):  
Pcr Amplification ◽  
Morphological Characters ◽  
Chili Pepper ◽  
Colletotrichum Acutatum ◽  
First Report ◽  
Blast Search ◽  
Rdna Its ◽  
Its Sequence Analysis ◽  
Pcr Products ◽  
Needle Prick

A new anthracnose disease on chili pepper (cayenne pepper cv. Hongxiu 2003, fruiting type pepper) was found in Zhijiang County, Hunan, China in 2009. The disease was observed only on the fruits. Lesions were generally elongated, on which dark acervuli were arranged concentrically. Later, cracking of older lesions was observed. With a microscope, fungal conidia were observed to be 15.8 × 4.1 μm, fusiform or oval with one end acute, and single celled with two to seven oil globules. No setae were found on the acervuli. Eight isolates (HNZJ001–HNZJ008) showed no difference in colony feature when cultured on potato dextrose agar. All the isolates showed white growth at the early stages, but colonies turned pink when they produced powdery spores and then finally became red gray. The average colony diameter was 68.5 to 72.3 mm after 7 days with obvious gray black concentric rings because of the development of aerial and substrate mycelia. After a needle-prick inoculation with a suspension of 1 × 106 spores per ml of HNZJ001 on 30 chili pepper fruits with three repeats, the same symptoms were observed and the same fungus was recovered. In bioassays, HNZJ001 caused lesions on both mature and immature fruits, while Glomerella cingulata strain LSQ1 (GenBank Accession No. HQ607386) used as a control did not infect immature fruits. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5. Sequencing of the PCR products of HNZJ001 (GenBank Accession No. GU059863) showed 100% identity to G. acutata (GenBank Accession No. EU008863) and Colletotrichum acutatum (GenBank Accession No. AF207794) after a BLAST search. The pathogen was identified as G. acutata (asexual stage: C. acutatum) on the basis of morphological characters and rDNA-ITS sequence analysis. Worldwide, it has been reported that pepper anthracnose might be caused by up to five species of Glomerella (Colletotrichum): G. cingulata, C. coccodes, C. capsici, C. dematium, and G. acutata (2), among which only the first three were previously reported in China. In recent years, G. acutata was reported on such plants as apple (3) and strawberry (1) in China, but not on pepper. To our knowledge, this is the first report of G. acutata on chili pepper in China. References: (1) X.-J. Ren et al. Acta Phytopathol. Sin. 38:325, 2008. (2) P. P. Than et al. Zhejiang Univ. Sci. B 9:764, 2008. (3) R. Zhang et al. Plant Dis. 92:1474, 2008.

scholarly journals Interference in PCR: Transcription amplifications of mixed PVY isolates

2010 ◽  
Vol 41 (No. 4) ◽  
pp. 125-131 ◽  
Author(s):  
A. Rosner ◽  
L. Maslenin
Keyword(s):  
Virus Infection ◽  
Virus Strain ◽  
Rna Synthesis ◽  
Virus Isolate ◽  
Pcr Amplification ◽  
Target Sequence ◽  
Specific Primers ◽  
Pcr Products ◽  
Amplification Process ◽  
Mixed Virus Infection

Interference in PCR and transcription amplifications by mixed virus templates is described. The yields of PCR amplification of closely related PVY isolates in a mixture were lower than those of the separate amplification of each individual virus strain, i.e. the two virus templates mutually interfered in their amplification process. When PCR products of mixed PVY isolates served as templates for transcription, RNA synthesis was also inhibited. This interference could be avoided by applying strain-specific primers in the separate amplification of each target sequence in the mixture. The use of strain-specific primers in PCR amplification of a mixed virus infection thus enabled detection of each virus isolate without interference.

scholarly journals DNA barcodes and new primers for nature’s pest controllers: the social wasps

Genome ◽  
2020 ◽  
Author(s):  
Ikechukwu Eugene Onah ◽  
Seirian Sumner
Keyword(s):  
Dna Barcode ◽  
Pcr Amplification ◽  
Social Wasps ◽  
Coi Gene ◽  
Dna Barcodes ◽  
Anthropogenic Pressures ◽  
Specific Primers ◽  
Prime Concern ◽  
The Social ◽  
Polistine Wasps

Globally, biodiversity is declining as a result of anthropogenic pressures, and this could lead to extinction of some species before they are discovered. The loss of insect taxa is of prime concern, given recent reports of significant declines in the populations of many taxa across the globe. Efforts to document biodiversity have met with several challenges, amongst which are the difficulties in using morphological features to discriminate species, especially in insects. DNA barcoding is a rapid and reliable method for species identification and discovery, but choosing appropriate primers to amplify the barcode region without coamplifying contaminants remains a key challenge. We developed and tested a set of primers for PCR amplification of the DNA barcode region of the COI gene in polistine wasps. We tested their efficacy in 36 species of vespid wasps, and the solitary wasp Zethus miniatus Saussure. Samples were obtained from Africa, Americas, Asia and Europe. The polistine-specific primers successfully amplified the barcode region for all polistines tested, without amplifying any Wolbachia present; they also worked with many species from the other Vespidae wasp subfamilies. The new primers are valuable for the discovery and accurate documentation of polistine wasps in the four continents.

scholarly journals Gaeumannomyces graminis vars. avenae, graminis, and tritici Identified Using PCR Amplification of Avenacinase-like Genes

Plant Disease ◽  
2002 ◽  
Vol 86 (6) ◽  
pp. 652-660 ◽  
Author(s):  
Sansanalak Rachdawong ◽  
Carole L. Cramer ◽  
Elizabeth A. Grabau ◽  
Verlyn K. Stromberg ◽  
George H. Lacy ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Rapid Identification ◽  
Gaeumannomyces Graminis ◽  
Specific Primers ◽  
Single Tube ◽  
Pcr Products ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Cereal Fields ◽  
Take All

Identifying take-all pathogens, Gaeumannomyces graminis varieties avenae (Gga), graminis (Ggg), and tritici (Ggt), is difficult. Rapid identification is important for development of disease thresholds. We developed a single-tube, polymerase chain reaction (PCR) method differentiating among Gga, Ggg, and Ggt. Nucleotide base sequence analyses of avenacinase-like genes from Gga, Ggg, and Ggt isolates provided the basis for designing variety-specific primers. Sequences from Ggg and Ggt were highly related (99% identity), but Gga sequences were <95% identical to Ggg and Ggt sequences. Three 5′ primers specific for Gga, Ggt, and Ggg and a single 3′ common primer allowed amplification of variety-specific fragments of 617, 870, and 1,086 bp, respectively. Each 5′ primer was specific in mixed populations of primers and templates. No PCR products were amplified from related fungi including Gaeumannomyces cylindrosporus and Phialophora spp. We surveyed 16 putative Ggt isolates using our assay; nine produced Ggt-specific fragments and seven produced Ggg-specific fragments. Five Gga isolates produced Gga-specific fragments. However, Gga- and Ggt-specific fragments were observed from a sixth Gga isolate, RB-W, which indicates a mixed culture or a heterokaryon. Our single-tube, PCR method rapidly differentiates among the important take-all pathogens commonly encountered together in cereal fields.

scholarly journals Presence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with reduced susceptibility to fluoroquinolones isolated from human salmonellosis in Gyeonggi-do, South Korea from 2016 to 2019

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sohyun Lee ◽  
Nanjoo Park ◽  
Sujung Yun ◽  
Eunseon Hur ◽  
Jiwon Song ◽  
...  
Keyword(s):  
South Korea ◽  
Pcr Amplification ◽  
Public Health Problem ◽  
Test Method ◽  
Quinolone Resistance ◽  
Human Salmonellosis ◽  
Pcr Products ◽  
Antimicrobial Susceptibility Tests ◽  
Susceptibility Tests ◽  
Sequence Types

AbstractNon-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene (qnrA, qnrB, or qnrS), and three Salmonella strains carried two PMQR genes (qnrS and aac(6′)-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,[5]12:i:- (32.4%, 11/34) and Salmonella Typhimurium (29.4%, 10/34) were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.

Organization, Expression and Evolution of a Disease Resistance Gene Cluster in Soybean

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 1961-1977
Author(s):  
Michelle A Graham ◽  
Laura Fredrick Marek ◽  
Randy C Shoemaker
Keyword(s):  
Disease Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Developmental Stages ◽  
Gene Evolution ◽  
Pcr Amplification ◽  
Disease Resistance Genes ◽  
Disease Resistance Gene ◽  
R Genes ◽  
Specific Primers

Abstract PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar “Williams 82” [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca2+-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron

Parasitology ◽  
2002 ◽  
Vol 124 (2) ◽  
pp. 177-184 ◽  
Author(s):  
M. B. A. MENDONÇA ◽  
N. S. NEHME ◽  
S. S. SANTOS ◽  
E. CUPOLILLO ◽  
N. VARGAS ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Ribosomal Rna ◽  
Southern Blotting ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Pcr Products ◽  
Phylogenetic Lineages ◽  
Definition Of ◽  
Ribosomal Cistron ◽  
Panstrongylus Geniculatus

Trypanosoma cruzi is currently classified into 2 major phylogenetic lineages, T. cruzi I and II, that correlate with the formerly described zymodeme 1 and 2, respectively. Another isoenzymic group (zymodeme 3–Z3) was also described. In this study, we analysed the genetic diversity among Z3 isolates of the Brazilian Amazon by restriction fragment length polymorphism of the intergenic transcribed spacers (ITSs) of the ribosomal RNA cistron and the size of the divergent domain D7 of the 24Sα rRNA gene. DNAs from 12 T. cruzi Z3 isolates obtained from humans (2), Panstrongylus geniculatus (1), and Rhodnius brethesi (9) were submitted to PCR amplification of the ITSs plus the 5·8S rDNA. The PCR products were digested with 4 distinct endonucleases and the profiles analysed by a numerical methodology. The phenetic dendrogram revealed a clear dichotomy in the Z3 group, defining 2 groups that were named Z3-A and Z3-B. Dimorphism was also found in the band sizes of the amplified D7 divergent domain of the 24Sα rDNA, which showed a perfect correlation with the ITSs clustering. The organization of the ribosomal cistron was investigated by Southern blotting and shown to be conserved in the genome of the 2 Z3 groups. This study shows that the rDNA cistron allows the definition of 2 distinct subclusters in Z3 isolates.

Variation in bacterial endosymbionts associated with the date palm hopper,Ommatissus lybicuspopulations

2017 ◽  
Vol 108 (2) ◽  
pp. 271-281 ◽  
Author(s):  
S. Karimi ◽  
H. Izadi ◽  
M. Askari Seyahooei ◽  
A. Bagheri ◽  
P. Khodaygan
Keyword(s):  
Date Palm ◽  
Multiple Infections ◽  
Infection Frequency ◽  
Pcr Assay ◽  
Specific Primers ◽  
Consensus Sequences ◽  
Pcr Products ◽  
Bacterial Endosymbionts ◽  
Different Populations ◽  
Polymerase Chain

AbstractThe date palm hopper,Ommatissus lybicus, is a key pest of the date palm, which is expected to be comprised of many allopatric populations. The current study was carried out to determine bacterial endosymbiont diversity in the different populations of this pest. Ten date palm hopper populations were collected from the main date palm growing regions in Iran and an additional four samples from Pakistan, Oman, Egypt and Tunisia for detection of primary and secondary endosymbionts using polymerase chain reaction (PCR) assay with their specific primers. The PCR products were directly sequenced and edited using SeqMan software. The consensus sequences were subjected to a BLAST similarity search. The results revealed the presence of ‘CandidatusSulcia muelleri’ (primary endosymbiont) andWolbachia,ArsenophonusandEnterobacter(secondary endosymbionts) in all populations. This assay failed to detect ‘CandidatusNasuia deltocephalinicola’ andSerratiain these populations. ‘Ca. S. muelleri’ exhibited a 100% infection frequency in populations andWolbachia,ArsenophonusandEnterobacterdemonstrated 100, 93.04 and 97.39% infection frequencies, respectively. The infection rate ofArsenophonusandEnterobacterranged from 75 to 100% and 62.5 to 100%, respectively, in different populations of the insect. The results demonstrated multiple infections by ‘Ca. Sulcia muelleri’,Wolbachia,ArsenophonusandEnterobacterin the populations and may suggest significant roles for these endosymbionts on date palm hopper population fitness. This study provides an insight to endosymbiont variation in the date palm hopper populations; however, further investigation is needed to examine how these endosymbionts may affect host fitness.

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