[Objective] In order to get a purified strain to carry out the relative molecular biology research about E.tenella. [Method] The single-oocyst isolation method was improved and the isolated single-oocyst which was put...[Objective] In order to get a purified strain to carry out the relative molecular biology research about E.tenella. [Method] The single-oocyst isolation method was improved and the isolated single-oocyst which was put into capsule was fed to chickens. At the same time, the collected oocysts were identified by PCR method. [Result] The oocysts were isolated from feces of 15 chickens among that of 20 chickens and the infection rate was 75%. The PCR results demonstrated that the single-oocyst strain was E.tenella. [Conclusion] The inoculation of single oocyst capsule was simple, besides, this method did not only save time but also declined inoculation difficulty, increased infection rate and provided good materials for biological research of coccidian.展开更多
The textile industrial chain all over the world is facing a challenge of differentiating cashmere fiber from mixture of wool and other fibers in case cashmere stocks are adulterated with wool or other fibers. For iden...The textile industrial chain all over the world is facing a challenge of differentiating cashmere fiber from mixture of wool and other fibers in case cashmere stocks are adulterated with wool or other fibers. For identification of cashmere in such mixtures, the development of microchip based real-time PCR technology offers a very sensitive, specific, and accurate solution. The technology has been validated with cashmere and wool samples procured from distant farms, and from cashmere goats and sheep of different age and sex. Model samples with incremental raw cashmere or wool content were tested. The experimentally determined content was found to be comparable to the weighed content of the respective fibers in the samples. This technology may prove a cost cutter since it needs only 1.2 μl of the PCR reagent mix. It is substantially faster than traditional real-time PCR systems for being carried as miniature reaction volume in metal microchip. These features allow faster thermal equilibrium and thermal uniformity over the entire array of microreactors. For routine tests or in commercial set up, the microchips are available as ready-to-run with lyophilized reagents in its microreactors to which only 1 μl of the 10-fold diluted isolated DNA sample is added. The lyophilized microchips offer user-friendly handling in testing laboratories and help minimize human error.展开更多
We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species.Ten reverse species-specific primers designed from the 16 S rRNA gene,in combination with one forward...We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species.Ten reverse species-specific primers designed from the 16 S rRNA gene,in combination with one forward universal primer,generated PCR fragments of ca.270 bp length for each species.The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species.Amplification was observed in specific species only.The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber,and was proven to be a useful,rapid,and low-cost technique to identify the origin of the sea cucumber product.展开更多
In this study, marine microplankton were identified by combining standard light microscopy with Sanger 18S rRNA gene sequencing. The image-matching individual PCR technique was applied to identify the image collectabl...In this study, marine microplankton were identified by combining standard light microscopy with Sanger 18S rRNA gene sequencing. The image-matching individual PCR technique was applied to identify the image collectable unicellular microplankton to genera. Instead of pure strain culture and morphological identification, microplankton individual cells were isolated and fixed with glutaraldehyde, frozen and stored for months. Finally, they were imaged under a microscope and molecularly identified via phylogenetic analysis of their 18S ribosomal RNA gene(18S rDNA). Microplankton cells were collected at 30 locations in South China Sea, and were assigned to 21 known and 4 unidentified genera(2 uncultured fungi and 2 uncultured stramenopiles) with phylogenetic analysis in parallel to the morphological identification.展开更多
Duck tembusu virus disease is one of the most serious infectious diseases endangering duck industry. A strain of virus was isolated from a dead duck,and performed PCR identification and sequence analysis. The results ...Duck tembusu virus disease is one of the most serious infectious diseases endangering duck industry. A strain of virus was isolated from a dead duck,and performed PCR identification and sequence analysis. The results showed that the sequence of the isolate shared above 99% homology with duck tembusu virus( DTMUV) sequence on Gen Bank. The result indicated that the isolated virus was DTMUV.展开更多
In order to establish a quick and specific method which could identify the avian-derived ingredients,this study used 16 S rRNA gene sequence as target site,and designed the specific primers of chicken,pigeon meat and ...In order to establish a quick and specific method which could identify the avian-derived ingredients,this study used 16 S rRNA gene sequence as target site,and designed the specific primers of chicken,pigeon meat and quail meat. The DNA of common livestock and poultry meat( including mutton,beef,pork,rabbit meat,pigeon meat,quail meat,chicken,duck and goose) was used as template. Though PCR amplification and specific detection,a quick determination method was established to identify the avian-derived ingredients. The results showed that the selected primers could identify the ingredients of animal origin effectively and quickly. The method was convenient and concise,and could detect the chicken-derived,pigeon-derived,quail-derived ingredients in livestock and poultry food quickly and accurately.展开更多
[Objective] The research aimed to distinguish Chinese herb Qingjiao from its botanical origin plants by comparing different DNA sequences,so as to provide a molecular basis for origin identification and quality evalua...[Objective] The research aimed to distinguish Chinese herb Qingjiao from its botanical origin plants by comparing different DNA sequences,so as to provide a molecular basis for origin identification and quality evaluation.[Method] The cpDNA psbA-trnH and nrDNA ITS sequences of five Chinese herb Qingjiao plants,including Gentiana macrophylla pall.,Gentiana straminea Maxim.,Gentiana crassicaulis Duthie ex Burk.,Gentiana dahurica Fisch and Gentiana officinalis H.Smith,were amplified with PCR,and then sequenced by direct PCR sequencing method for homologous analysis.[Results] The length of cpDNA psbA-trnH of five plants was 316-318 bp;there were seven different haplotypes and seven variable sites;the GC content of the sequence was 21.2%;the phylogenetic clustering showed the same result as haplotype analysis.The length of nrDNA ITS sequence of five plants was 624-625 bp,there were five different haplotypes and 13 variable sites;the GC content of the sequence was 59.3%.The result of phylogenetic clustering suggested that G.dahurica and G.straminea,G.macrophylla and G.officinalis clustered together as sister clades,respectively.[Conclusion] The nucleotide differences of nrDNA ITS regions could be used for distinguishing botanical origin in Chinese herb Qingjiao.展开更多
Herba Anoectochili is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the similar morphological characteristics of Herba Anoectochili and its adulterants, traditional identif...Herba Anoectochili is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the similar morphological characteristics of Herba Anoectochili and its adulterants, traditional identification techniques often fail to distinguish between them accurately, which is not conducive to the circulation management and safety of the medicinal materials. To improve the distinction between Herba Anoectochili and its adulterants accurately, this study identified 41 Herba Anoectochili and its adulterant samples based on the ITS2 sequence. Sequence characteristics, Basic Local Alignment Search Tool(BLAST) application, genetic distance, construction of phylogenetic tree, secondary structure prediction, and other methods showed the ITS2 sequence to accurately identify Herba Anoectochili from its adulterants. Furthermore, in this study, we designed a specific primer, based on the ITS2 sequence, and established a real-time PCR detection system for the rapid, sensitive, and specific identification of the original plant of Herba Anoectochili. Compared to DNA barcoding technology, this method has shorter detection time, stronger specificity, and higher sensitivity, which lays the foundation for the rapid identification of Herba Anoectochili.展开更多
Plant-parasitic nematodes are very common on cereal crops and cause economic losses via reduction in grain quality and quantity. During 2014, 83 soil samples were collected from wheat and barley fields in 21 districts...Plant-parasitic nematodes are very common on cereal crops and cause economic losses via reduction in grain quality and quantity. During 2014, 83 soil samples were collected from wheat and barley fields in 21 districts of 13 provinces across five regions （CentralAnatolia, Marmara, Aegean, SoutheastAnatolia, and Black Sea Region） of Turkey. Cyst-forming nematodes were found in 66 samples （80%）, and the internal transcribed spacer （ITS） sequencing and species-specific PCR identified the species in 64 samples as Heterodera filipjevi, Heterodera latipons, and Heterodera avenae. The predominant patho- genic cereal cyst nematode was H. filipjevi, which was found in all five regions surveyed. H. avenae was only detected in Southeast Anatolia whereas H. latipons was detected in Southeast Anatolia and Central Anatolia. ITS-rDNA phylogenetic analyses showed that H. avenae isolates from China clustered with H. australis, and Turkish isolates were closely related to European and USA isolates of this species. H. filipjevi from Turkey and China were clustered closely with those from the UK, Germany, Russia, and the USA. The density of many of these populations exceeded 6r approached the maximum threshold level for economic loss. To our knowledge, this is the first report of H. filipjevi in Diyarbakir, Edirne, and Kutahya provinces, and the first report of H. avenae in DiyarbakJr Province. These results exhibit the most rigorous analysis to date on the occurrence and distribution of Heterodera spp. in Turkey＇s major wheat-producing areas, thus providing a basis for more specific resistance breeding, as well as other management practices.展开更多
Death of infants from diarrhoea is a common occurrence in sub-Saharan Africa. This is attributed to unhygienic practices which aid the proliferation of diarrhoea-causing microorganisms. Among these microorganisms, Cam...Death of infants from diarrhoea is a common occurrence in sub-Saharan Africa. This is attributed to unhygienic practices which aid the proliferation of diarrhoea-causing microorganisms. Among these microorganisms, Cam- pylobacter species have been reported as one of the causal agents, Campylobacter spp. are human intestinal pathogens of global importance and their pathogenicity mechanisms are not well understood. This study was designed to investigate the molecular characterisation of Campylobacter gotten from cultural methods in Osun State. Campylobacters isolated were biochemically characterized and biotyped. Confirmation of Campylobacter was done using flaA gene, hippuricase O for Campylobacter jejuni and aspartokinase gene for Campylobacter coli and single locus sequencing glnA gene were performed by PCR. Twenty five samples were amplified by PCR out of 57 Campylobacter strains that were positive for cultural methods from 815 stool samples with diarrhoea and 100 stool samples without diarrhoea. No Campylobacter was isolated from stools of children in the control group. Twenty-five isolates comprising of 18 Campylobater jejuni and 7 C. coli were identified. The nucleotide sequence of the gln A for all the isolated Campylobacter spp. showed 91.0% similarity with the ones in the GenBank. The C. jejuni was classified into biotypes I (44.4%) and II (55.6%) and all C. coli were of biotype I.展开更多
Pasteurella species is considered the principal pathogen of the respiratory tract.Mannheimia haemolytica and Pasteurella multocida were investigated and typed from nasal swabs and tissues taken from sheep,goat and cat...Pasteurella species is considered the principal pathogen of the respiratory tract.Mannheimia haemolytica and Pasteurella multocida were investigated and typed from nasal swabs and tissues taken from sheep,goat and cattle.Indeed,41 lung and 121 nasal swabs samples were collected from animals with respiratory diseases during 2015 to 2017 in six different regions in Morocco.At first,a screening of Pasteurella species using the real time PCR(RT-PCR)was carried out,then all isolated strains on agar blood were confirmed by PCR gel based assay specific for M.haemolytica and P.multocida.Pathogenicity was evaluated in mice and histopathological examination was done on some of lung tissue.The results revealed that 34 samples of which 28(55%)from nasal swabs and six(38%)from lungs were positive for M.haemolytica and nine samples of which seven(14%)from nasal swabs and two(13%)from lungs were positive on P.multocida serogroup A.Seventy-two percent(72%)isolates were highly pathogenic to mice,which is in accordance with the results obtained by histopathology examination.This is the first report for widespread infections of Pasteurella(M.haemolytica&P.multocida)in ruminants in Morocco.Therefore,measures including development of vaccines are highly required to mitigate the impact of the bacteria in animals.展开更多
Rotted potato samples were collected from ventilated storage house of Huade County,Inner Mongolia from October 2015 to April 2016. Pathogens were isolated,purified and cultured with tissue separation method,and identi...Rotted potato samples were collected from ventilated storage house of Huade County,Inner Mongolia from October 2015 to April 2016. Pathogens were isolated,purified and cultured with tissue separation method,and identified by morphological observation and molecular identification. The results showed that there were five main diseases that caused potato rotting in Huande County,Inner Mongolia during the storage period,including dry rot,late blight,early blight,ring rot and soft rot. Specifically,potato dry rot was the most serious disease,which was induced by four pathogens: Fusarium sambucinum,Fusarium Link,Fusarium acuminatum and Fusarium equiseti. Late blight caused by Phytophthora infestans and early blight caused by Alternaria solani could easily result in complex infection,which were mostly found in seriously rotted potato tubers. Moreover,these potatoes were always co-infected with fungal diseases at late stage of storage or even subject to complex infection with bacteriosis in serious cases. This study provided theoretical basis for the prevention of potato diseases during the storage period.展开更多
In this study, a rapid molecular identification method of Tribolium destructor was established with PCR and PCR-RFLP （polymerase chain reaction-restriction fragment length polymorphism） technology. According to the ...In this study, a rapid molecular identification method of Tribolium destructor was established with PCR and PCR-RFLP （polymerase chain reaction-restriction fragment length polymorphism） technology. According to the results, （ 1 ） with PCR method, specific primers were designed based on CO1 gene of T. destructor for PCR amplification, and electrophoresis detection confirmed that PCR method could be used to rapidly and accurately identify T. destructor; （2） with PCR-RFLP method, two pairs of degenerate primers were used to amplify CO1 gene of Tribolium species, PCR products were digested with HindIII and detected by electrophoresis, results indicated that PCR-RFLP method could also be used for rapid identification of T. destructor in quarantine practice.展开更多
Effect of different Zinc doses was investigated against Erwinia carotovora ssp. atroseptica, the potato blackleg/soft rot causing organism, during 2009 and 2010 in Department of Plant Pathology and Institute of Biotec...Effect of different Zinc doses was investigated against Erwinia carotovora ssp. atroseptica, the potato blackleg/soft rot causing organism, during 2009 and 2010 in Department of Plant Pathology and Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar-Pakistan. Out of 200 tested samples, 21 of them were proved to be Eca. However, these tentative Eca isolates showed some characteristics which were unexpected for Eca. We, therefore, decided to perform Polymerase Chain Reaction using Eca-specific primers, Eca1F and Eca2R for confirm identification. For disease management, at the time of sowing, pots containing 5 kg sterilized soil were applied with Zinc in four different treatments i.e. 8 mg, 10 mg, 12 mg and 14 mg along with one control. Results indicated that 12 mg (4.8 kg Zn ha-1) were better doses in controlling the disease up to 73% and increasing the yield up to 117% as compared to control plants.展开更多
A bacterial strain was isolated from the sick pigs suspiciously infected by polyserositis and arthritis in a pig farm in Shandong Province, and identified through morphological observation, culture traits, bioehemical...A bacterial strain was isolated from the sick pigs suspiciously infected by polyserositis and arthritis in a pig farm in Shandong Province, and identified through morphological observation, culture traits, bioehemical characteristics and PCR amplifieation. Additionally, primers were de- signed according to the 16S rRNA sequence of Haemophihzs parasuis, and the bacterial strain was amplified by PCR. The amplified fragments of approximately 1 400 bp was sequenced, and aligned with the sequence in GenBank. The results showed that it shared the homology of 97%-99% with the 16S rRNA sequence of foreign H.parasuis, and confirmed as H.parasuis （HPS）. The strain was determined as serotype 4 through serotype identification. The strain was named SD02.展开更多
The diagnosis of bacterial or fungal infections requires the identification of the pathogen etiology in the shortest time possible. Although some biomarkers are used as indicators of bacterial infections, their specif...The diagnosis of bacterial or fungal infections requires the identification of the pathogen etiology in the shortest time possible. Although some biomarkers are used as indicators of bacterial infections, their specificity and sensitivity are highly variable, and there is no direct relationship between the level increase of these biomarkers for mycosis. It is common to obtain negative microbiological cultures in patients infected by non-culturable, intracellular bacteria or mycosis, even though there is a high clinical suspicion of infection. This study identifies the pathogen present in critically infected patients through 16S and 18S/eEF1 genes detection by polymerase chain reaction (PCR) coupled with Sanger sequencing. Thirty clinical samples were evaluated by PCR, of which 40% were positive for fungi, 23.33% for bacteria, 26.7% for fungi and bacteria, and 10% for no pathogen. The PCRs outcomes period for bacteria or fungi was one day compared to seven and up to 14 days (on average) of microbiological culture for bacteria and fungi. Then, we assessed the relationship with the most used biomarkers (procalcitonin, C-reactive protein, globular sedimentation velocity, and the neutrophil-lymphocyte index). This combination of molecular techniques has been shown as helpful in identifying intracellular bacteria and fungi that are difficult to culture by conventional methods. Screening with genomic markers 16S and 18S/eEF1 by PCR allowed us to optimize the time to obtain the result of the infection caused by bacteria or fungi. Also, identifying the specific etiological microorganism by Sanger sequencing was very helpful in avoiding the progression of the disease and setting targeted treatment with better clinical outcomes.展开更多
[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 f...[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.展开更多
[Objectives] To identify ITS2 barcode of Lablab Semen Album and its adulterants,and provide a new method for the identification of Lablab Semen Album. [Methods] The ITS2 sequence was amplified by PCR and sequenced bi-...[Objectives] To identify ITS2 barcode of Lablab Semen Album and its adulterants,and provide a new method for the identification of Lablab Semen Album. [Methods] The ITS2 sequence was amplified by PCR and sequenced bi-directionally. After splicing by Codon Code Aligner,the data were processed with the aid MEGA software to construct the cluster dendrogram( neighbor-joining,NJ tree). [Results]The ITS2 sequence of Lablab Semen Album had length of 218 bp; the constructed cluster dendrogram indicated that all species were monophyletic and could be distinguished from other species. [Conclusions] The ITS2 barcode can be used for rapid identification of Lablab Semen Album and its adulterants and this experiment further verified that DNA barcode technology is effective in identification of traditional Chinese medicines.展开更多
Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania specie...Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.Methods:A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.Results:This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.Conclusions:Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.展开更多
基金Supported by the National Natural Science Foundation of China(30671580, 30170696)~~
文摘[Objective] In order to get a purified strain to carry out the relative molecular biology research about E.tenella. [Method] The single-oocyst isolation method was improved and the isolated single-oocyst which was put into capsule was fed to chickens. At the same time, the collected oocysts were identified by PCR method. [Result] The oocysts were isolated from feces of 15 chickens among that of 20 chickens and the infection rate was 75%. The PCR results demonstrated that the single-oocyst strain was E.tenella. [Conclusion] The inoculation of single oocyst capsule was simple, besides, this method did not only save time but also declined inoculation difficulty, increased infection rate and provided good materials for biological research of coccidian.
文摘The textile industrial chain all over the world is facing a challenge of differentiating cashmere fiber from mixture of wool and other fibers in case cashmere stocks are adulterated with wool or other fibers. For identification of cashmere in such mixtures, the development of microchip based real-time PCR technology offers a very sensitive, specific, and accurate solution. The technology has been validated with cashmere and wool samples procured from distant farms, and from cashmere goats and sheep of different age and sex. Model samples with incremental raw cashmere or wool content were tested. The experimentally determined content was found to be comparable to the weighed content of the respective fibers in the samples. This technology may prove a cost cutter since it needs only 1.2 μl of the PCR reagent mix. It is substantially faster than traditional real-time PCR systems for being carried as miniature reaction volume in metal microchip. These features allow faster thermal equilibrium and thermal uniformity over the entire array of microreactors. For routine tests or in commercial set up, the microchips are available as ready-to-run with lyophilized reagents in its microreactors to which only 1 μl of the 10-fold diluted isolated DNA sample is added. The lyophilized microchips offer user-friendly handling in testing laboratories and help minimize human error.
基金Supported by the National Natural Science Foundation of China(No.31201999)the Natural Science Foundation of Guangdong Province,China(No.S2011040000463)+4 种基金the Foundation for Distinguished Young Talents in Higher Education of Guangdong,China(No.LYM11086)the Key Laboratory Program of Tropical Marine Bio-Resources and Ecology,Chinese Academy of Science(No.LMB111004)the China Spark Program(Nos.2012GA780007,2012GA780020,2012GA780008)the National Students'Innovation and Entrepreneurship Training Project(No.201210579031)the Zhanjiang Foundation for Science and Technology,China(Nos.2011C3104009,2011D0244,2012C3102018)
文摘We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species.Ten reverse species-specific primers designed from the 16 S rRNA gene,in combination with one forward universal primer,generated PCR fragments of ca.270 bp length for each species.The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species.Amplification was observed in specific species only.The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber,and was proven to be a useful,rapid,and low-cost technique to identify the origin of the sea cucumber product.
基金supported by Hebei Province Natural Science Foundation for Youths (No. C2015202202)Colleges and Universities in Hebei Province Science and Technology Research Project (No. QN20131082)+1 种基金the National Natural Science Foundation of China (No. 51474084)the National Key Research and Development Program of China (No. 2016YFB0601001)
文摘In this study, marine microplankton were identified by combining standard light microscopy with Sanger 18S rRNA gene sequencing. The image-matching individual PCR technique was applied to identify the image collectable unicellular microplankton to genera. Instead of pure strain culture and morphological identification, microplankton individual cells were isolated and fixed with glutaraldehyde, frozen and stored for months. Finally, they were imaged under a microscope and molecularly identified via phylogenetic analysis of their 18S ribosomal RNA gene(18S rDNA). Microplankton cells were collected at 30 locations in South China Sea, and were assigned to 21 known and 4 unidentified genera(2 uncultured fungi and 2 uncultured stramenopiles) with phylogenetic analysis in parallel to the morphological identification.
基金Supported by National Natural Science Foundation of China(31402224)Key Research & Development Project of Hainan Province(ZDYF2017029)Agricultural Science and Technology Innovation Project of Hainan Academy of Agricultural Sciences(CXZX201413)
文摘Duck tembusu virus disease is one of the most serious infectious diseases endangering duck industry. A strain of virus was isolated from a dead duck,and performed PCR identification and sequence analysis. The results showed that the sequence of the isolate shared above 99% homology with duck tembusu virus( DTMUV) sequence on Gen Bank. The result indicated that the isolated virus was DTMUV.
基金Supported by New Agricultural Variety,Technology and Model Project in Jiangsu Province(SXGC2015298)Project on Prospective Study of Social Development in Yangzhou City(YZ2014188)+1 种基金Science and Technology Public Service Platform Construction Project in Yangzhou City(YZ2015162)National Agricultural Product Quality and Safety Risk Assessment Project in 2016(GJFP2016007)
文摘In order to establish a quick and specific method which could identify the avian-derived ingredients,this study used 16 S rRNA gene sequence as target site,and designed the specific primers of chicken,pigeon meat and quail meat. The DNA of common livestock and poultry meat( including mutton,beef,pork,rabbit meat,pigeon meat,quail meat,chicken,duck and goose) was used as template. Though PCR amplification and specific detection,a quick determination method was established to identify the avian-derived ingredients. The results showed that the selected primers could identify the ingredients of animal origin effectively and quickly. The method was convenient and concise,and could detect the chicken-derived,pigeon-derived,quail-derived ingredients in livestock and poultry food quickly and accurately.
基金Supported by Science Foundation for the Excellent Youth and Middle-aged Scholars in Qinghai University(2009-QY-19)~~
文摘[Objective] The research aimed to distinguish Chinese herb Qingjiao from its botanical origin plants by comparing different DNA sequences,so as to provide a molecular basis for origin identification and quality evaluation.[Method] The cpDNA psbA-trnH and nrDNA ITS sequences of five Chinese herb Qingjiao plants,including Gentiana macrophylla pall.,Gentiana straminea Maxim.,Gentiana crassicaulis Duthie ex Burk.,Gentiana dahurica Fisch and Gentiana officinalis H.Smith,were amplified with PCR,and then sequenced by direct PCR sequencing method for homologous analysis.[Results] The length of cpDNA psbA-trnH of five plants was 316-318 bp;there were seven different haplotypes and seven variable sites;the GC content of the sequence was 21.2%;the phylogenetic clustering showed the same result as haplotype analysis.The length of nrDNA ITS sequence of five plants was 624-625 bp,there were five different haplotypes and 13 variable sites;the GC content of the sequence was 59.3%.The result of phylogenetic clustering suggested that G.dahurica and G.straminea,G.macrophylla and G.officinalis clustered together as sister clades,respectively.[Conclusion] The nucleotide differences of nrDNA ITS regions could be used for distinguishing botanical origin in Chinese herb Qingjiao.
基金supported by the National Key Research and Development Program "Chinese Medicine Modernization Research" Key Project(No.2017YFC1702100)Wuhan University of Technology Professional Degree Graduate Team Guidance Project(No.201701)Wuhan University of Technology Graduate Public Experimental Course Construction Project(No.201906)
文摘Herba Anoectochili is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the similar morphological characteristics of Herba Anoectochili and its adulterants, traditional identification techniques often fail to distinguish between them accurately, which is not conducive to the circulation management and safety of the medicinal materials. To improve the distinction between Herba Anoectochili and its adulterants accurately, this study identified 41 Herba Anoectochili and its adulterant samples based on the ITS2 sequence. Sequence characteristics, Basic Local Alignment Search Tool(BLAST) application, genetic distance, construction of phylogenetic tree, secondary structure prediction, and other methods showed the ITS2 sequence to accurately identify Herba Anoectochili from its adulterants. Furthermore, in this study, we designed a specific primer, based on the ITS2 sequence, and established a real-time PCR detection system for the rapid, sensitive, and specific identification of the original plant of Herba Anoectochili. Compared to DNA barcoding technology, this method has shorter detection time, stronger specificity, and higher sensitivity, which lays the foundation for the rapid identification of Herba Anoectochili.
基金financially supported by the Special Fund for Agro-scientific Research in the Public Interest,China(201503114 and 200903040)the National Key Basic Research Program of China(973 Program,2013CB127502)
文摘Plant-parasitic nematodes are very common on cereal crops and cause economic losses via reduction in grain quality and quantity. During 2014, 83 soil samples were collected from wheat and barley fields in 21 districts of 13 provinces across five regions （CentralAnatolia, Marmara, Aegean, SoutheastAnatolia, and Black Sea Region） of Turkey. Cyst-forming nematodes were found in 66 samples （80%）, and the internal transcribed spacer （ITS） sequencing and species-specific PCR identified the species in 64 samples as Heterodera filipjevi, Heterodera latipons, and Heterodera avenae. The predominant patho- genic cereal cyst nematode was H. filipjevi, which was found in all five regions surveyed. H. avenae was only detected in Southeast Anatolia whereas H. latipons was detected in Southeast Anatolia and Central Anatolia. ITS-rDNA phylogenetic analyses showed that H. avenae isolates from China clustered with H. australis, and Turkish isolates were closely related to European and USA isolates of this species. H. filipjevi from Turkey and China were clustered closely with those from the UK, Germany, Russia, and the USA. The density of many of these populations exceeded 6r approached the maximum threshold level for economic loss. To our knowledge, this is the first report of H. filipjevi in Diyarbakir, Edirne, and Kutahya provinces, and the first report of H. avenae in DiyarbakJr Province. These results exhibit the most rigorous analysis to date on the occurrence and distribution of Heterodera spp. in Turkey＇s major wheat-producing areas, thus providing a basis for more specific resistance breeding, as well as other management practices.
文摘Death of infants from diarrhoea is a common occurrence in sub-Saharan Africa. This is attributed to unhygienic practices which aid the proliferation of diarrhoea-causing microorganisms. Among these microorganisms, Cam- pylobacter species have been reported as one of the causal agents, Campylobacter spp. are human intestinal pathogens of global importance and their pathogenicity mechanisms are not well understood. This study was designed to investigate the molecular characterisation of Campylobacter gotten from cultural methods in Osun State. Campylobacters isolated were biochemically characterized and biotyped. Confirmation of Campylobacter was done using flaA gene, hippuricase O for Campylobacter jejuni and aspartokinase gene for Campylobacter coli and single locus sequencing glnA gene were performed by PCR. Twenty five samples were amplified by PCR out of 57 Campylobacter strains that were positive for cultural methods from 815 stool samples with diarrhoea and 100 stool samples without diarrhoea. No Campylobacter was isolated from stools of children in the control group. Twenty-five isolates comprising of 18 Campylobater jejuni and 7 C. coli were identified. The nucleotide sequence of the gln A for all the isolated Campylobacter spp. showed 91.0% similarity with the ones in the GenBank. The C. jejuni was classified into biotypes I (44.4%) and II (55.6%) and all C. coli were of biotype I.
文摘Pasteurella species is considered the principal pathogen of the respiratory tract.Mannheimia haemolytica and Pasteurella multocida were investigated and typed from nasal swabs and tissues taken from sheep,goat and cattle.Indeed,41 lung and 121 nasal swabs samples were collected from animals with respiratory diseases during 2015 to 2017 in six different regions in Morocco.At first,a screening of Pasteurella species using the real time PCR(RT-PCR)was carried out,then all isolated strains on agar blood were confirmed by PCR gel based assay specific for M.haemolytica and P.multocida.Pathogenicity was evaluated in mice and histopathological examination was done on some of lung tissue.The results revealed that 34 samples of which 28(55%)from nasal swabs and six(38%)from lungs were positive for M.haemolytica and nine samples of which seven(14%)from nasal swabs and two(13%)from lungs were positive on P.multocida serogroup A.Seventy-two percent(72%)isolates were highly pathogenic to mice,which is in accordance with the results obtained by histopathology examination.This is the first report for widespread infections of Pasteurella(M.haemolytica&P.multocida)in ruminants in Morocco.Therefore,measures including development of vaccines are highly required to mitigate the impact of the bacteria in animals.
基金Supported by National Key Research and Development Program of China"Research and Demonstration of Derogation Technical Equipment and Model for Postpartum Storage and Transportation of Potato Tubers"(2016YFD0401301)
文摘Rotted potato samples were collected from ventilated storage house of Huade County,Inner Mongolia from October 2015 to April 2016. Pathogens were isolated,purified and cultured with tissue separation method,and identified by morphological observation and molecular identification. The results showed that there were five main diseases that caused potato rotting in Huande County,Inner Mongolia during the storage period,including dry rot,late blight,early blight,ring rot and soft rot. Specifically,potato dry rot was the most serious disease,which was induced by four pathogens: Fusarium sambucinum,Fusarium Link,Fusarium acuminatum and Fusarium equiseti. Late blight caused by Phytophthora infestans and early blight caused by Alternaria solani could easily result in complex infection,which were mostly found in seriously rotted potato tubers. Moreover,these potatoes were always co-infected with fungal diseases at late stage of storage or even subject to complex infection with bacteriosis in serious cases. This study provided theoretical basis for the prevention of potato diseases during the storage period.
基金Supported by Science and Technology Project of Jiangsu Entry and Exit Inspection and Quarantine Bureau(2010KJ06)
文摘In this study, a rapid molecular identification method of Tribolium destructor was established with PCR and PCR-RFLP （polymerase chain reaction-restriction fragment length polymorphism） technology. According to the results, （ 1 ） with PCR method, specific primers were designed based on CO1 gene of T. destructor for PCR amplification, and electrophoresis detection confirmed that PCR method could be used to rapidly and accurately identify T. destructor; （2） with PCR-RFLP method, two pairs of degenerate primers were used to amplify CO1 gene of Tribolium species, PCR products were digested with HindIII and detected by electrophoresis, results indicated that PCR-RFLP method could also be used for rapid identification of T. destructor in quarantine practice.
文摘Effect of different Zinc doses was investigated against Erwinia carotovora ssp. atroseptica, the potato blackleg/soft rot causing organism, during 2009 and 2010 in Department of Plant Pathology and Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar-Pakistan. Out of 200 tested samples, 21 of them were proved to be Eca. However, these tentative Eca isolates showed some characteristics which were unexpected for Eca. We, therefore, decided to perform Polymerase Chain Reaction using Eca-specific primers, Eca1F and Eca2R for confirm identification. For disease management, at the time of sowing, pots containing 5 kg sterilized soil were applied with Zinc in four different treatments i.e. 8 mg, 10 mg, 12 mg and 14 mg along with one control. Results indicated that 12 mg (4.8 kg Zn ha-1) were better doses in controlling the disease up to 73% and increasing the yield up to 117% as compared to control plants.
文摘A bacterial strain was isolated from the sick pigs suspiciously infected by polyserositis and arthritis in a pig farm in Shandong Province, and identified through morphological observation, culture traits, bioehemical characteristics and PCR amplifieation. Additionally, primers were de- signed according to the 16S rRNA sequence of Haemophihzs parasuis, and the bacterial strain was amplified by PCR. The amplified fragments of approximately 1 400 bp was sequenced, and aligned with the sequence in GenBank. The results showed that it shared the homology of 97%-99% with the 16S rRNA sequence of foreign H.parasuis, and confirmed as H.parasuis （HPS）. The strain was determined as serotype 4 through serotype identification. The strain was named SD02.
文摘The diagnosis of bacterial or fungal infections requires the identification of the pathogen etiology in the shortest time possible. Although some biomarkers are used as indicators of bacterial infections, their specificity and sensitivity are highly variable, and there is no direct relationship between the level increase of these biomarkers for mycosis. It is common to obtain negative microbiological cultures in patients infected by non-culturable, intracellular bacteria or mycosis, even though there is a high clinical suspicion of infection. This study identifies the pathogen present in critically infected patients through 16S and 18S/eEF1 genes detection by polymerase chain reaction (PCR) coupled with Sanger sequencing. Thirty clinical samples were evaluated by PCR, of which 40% were positive for fungi, 23.33% for bacteria, 26.7% for fungi and bacteria, and 10% for no pathogen. The PCRs outcomes period for bacteria or fungi was one day compared to seven and up to 14 days (on average) of microbiological culture for bacteria and fungi. Then, we assessed the relationship with the most used biomarkers (procalcitonin, C-reactive protein, globular sedimentation velocity, and the neutrophil-lymphocyte index). This combination of molecular techniques has been shown as helpful in identifying intracellular bacteria and fungi that are difficult to culture by conventional methods. Screening with genomic markers 16S and 18S/eEF1 by PCR allowed us to optimize the time to obtain the result of the infection caused by bacteria or fungi. Also, identifying the specific etiological microorganism by Sanger sequencing was very helpful in avoiding the progression of the disease and setting targeted treatment with better clinical outcomes.
基金Supported by Natural Science Foundation of Fujian Province (2011J01066, 2012JO1061)。
文摘[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.
基金Supported by Key Program for Sci-tech Plan of Hunan Province Science and Technology Department(2014SK2001)Sci-tech Project for Food and Drug Safety of Hunan Food and Drug Administration(Xiang Shi Yao Ke R201612)
文摘[Objectives] To identify ITS2 barcode of Lablab Semen Album and its adulterants,and provide a new method for the identification of Lablab Semen Album. [Methods] The ITS2 sequence was amplified by PCR and sequenced bi-directionally. After splicing by Codon Code Aligner,the data were processed with the aid MEGA software to construct the cluster dendrogram( neighbor-joining,NJ tree). [Results]The ITS2 sequence of Lablab Semen Album had length of 218 bp; the constructed cluster dendrogram indicated that all species were monophyletic and could be distinguished from other species. [Conclusions] The ITS2 barcode can be used for rapid identification of Lablab Semen Album and its adulterants and this experiment further verified that DNA barcode technology is effective in identification of traditional Chinese medicines.
文摘Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.Methods:A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.Results:This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.Conclusions:Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.
