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1.
Anal Chem ; 92(1): 830-837, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31762266

RESUMO

The accurate and rapid monitoring of the expression levels of enterovirus 71 (EV71)-related microRNAs (miRNAs) can contribute to diagnosis of hand, foot, and mouth disease (HFMD) at the early stage. However, there is currently a lack of convenient methods for simultaneous monitoring of multiplex miRNAs in one step. Herein a one-step method for the simultaneous monitoring of multiple EV71 infection-related miRNAs is developed based on core-satellite structure assembled with magnetic nanobeads and quantum dots (MNs-ssDNA-QDs). In the presence of target miRNAs, duplex-specific nuclease (DSN)-assisted target recycling can be triggered, resulting in the release of QDs and recycling of target miRNAs. Then the simultaneous quantification can be easily realized by recording the corresponding amplified fluorescence signal of QDs in the suspension. With this method, simultaneous detection of hsa-miRNA-296-5p and hsa-miRNA-16-5p, potential biomarkers of EV71 infection, can be easily achieved with femtomolar sensitivity and single-base mismatch specificity. Moreover, the method is successfully used for monitoring of the expression level of miRNAs in EV71-infected cells at different time points, demonstrating the potential for diagnostic applications. With the merits of one-step operation and single-nucleotide mismatch discrimination, this work opens a new avenue for multiplex miRNAs detection. As different nucleotide sequences and multicolor QDs can be employed, this work is expected to offer great potential for the development of high throughput diagnosis.


Assuntos
Enterovirus Humano A/fisiologia , Infecções por Enterovirus/genética , Interações Hospedeiro-Patógeno , MicroRNAs/genética , Pontos Quânticos/química , Biomarcadores/análise , Linhagem Celular , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Infecções por Enterovirus/diagnóstico , Regulação da Expressão Gênica , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Nanopartículas de Magnetita/química , MicroRNAs/análise , Espectrometria de Fluorescência/métodos
2.
Analyst ; 145(6): 2184-2190, 2020 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-32101227

RESUMO

The continuing hurdle of developing foodborne pathogen detection techniques is that compromises must be made among simplicity, portability, speed, sensitivity, and quantitation. Herein, we fabricated quantum dot nanobeads (QDNS) by a layer-by-layer assembly of quantum dots on the surface of polymer nanospheres. QDNS exhibited higher fluorescence intensity than the quantum dots at the same particle number. Based on the quantum dot nanobeads as the signal reporter, a quantitative lateral flow immunoassay was demonstrated for Salmonella typhimurium detection with improved sensitivity, specificity and accuracy. A visual detection limit of 5 × 103 CFU mL-1Salmonella typhimurium within 10 min has been proved and demonstrated. Additionally, higher concentrations of non-Salmonella typhimurium bacteria have negligible effects on the detection of Salmonella typhimurium. The results of 50 single blind tests by 10 testers suggested that the assay exhibited 100% accuracy. The results illustrate that the assay provides a balance among simplicity, speed, sensitivity and accuracy, and it can be a favorable alternative for Salmonella typhimurium screening in various samples.


Assuntos
Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Pontos Quânticos/química , Salmonella typhimurium/isolamento & purificação , Resinas Acrílicas/química , Anticorpos Imobilizados/imunologia , Cádmio/química , Limite de Detecção , Nanosferas/química , Poliestirenos/química , Reprodutibilidade dos Testes , Salmonella typhimurium/imunologia , Selênio/química , Método Simples-Cego , Sulfetos/química , Compostos de Zinco/química
3.
Anal Chem ; 91(1): 1178-1184, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30516043

RESUMO

Rapid and sensitive foodborne pathogen detection assay, which can be applied in multiple fields, is essential to timely diagnosis. Herein, we proposed a multisignal readout lateral flow immunoassay for Salmonella typhimurium ( S. typhi) detection. The assay employs colorimetric-fluorescent-magnetic nanospheres (CFMNs) as labels, which possess multifunctional target separation and enrichment, multisignal readout, and two formats of quantitation. The assay for S. typhi detection involves magnetic separation and chromatography. First, the S. typhi were separated and enriched from matrix by antibody labeled CFMNs, and then the S. typhi-containing suspension is added onto the sample pad to flow up the test strip. The introduction of magnetic separation enhances anti-interference ability and 10-fold sensitivity, making the assay possible for practical application. The assay has realized naked eye detection of 1.88 × 104 CFU/mL S. typhi, and 3.75 × 103 CFU/mL S. typhi can be detected with a magnetic assay reader, which is 2-4 orders of magnitude lower than other label-based LFIAs, with a quantitation range of 1.88 × 104 to 1.88 × 107 CFU/mL by measuring the fluorescence intensity and magnetic signal. Moreover, the successful detection of S. typhi in complex matrix (tap water, milk, fetal bovine serum, and whole blood) indicated its potential application in real samples.


Assuntos
Colorimetria , Fluorescência , Nanopartículas de Magnetita/química , Salmonella typhimurium/isolamento & purificação , Microbiologia de Alimentos
4.
Anal Chem ; 91(11): 7280-7287, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31050409

RESUMO

The rapid and sensitive detection of pathogens is extremely crucial for timely clinical diagnosis and diseases control. Here, by employing cellular beacons with in situ synthesized QDs created from Staphylococcus aureus ( S. aureus), we efficiently fabricated an antibody (Ab) and acetylcholinesterase (AChE)-functionalized nanobioprobe, i.e., multifunctional cellular beacons (MCBs), avoiding complicated modification. Coupled with magnetic separation, a novel method for pathogen detection with the naked eye is established. With this method, enterovirus 71 (EV71) can be detected by the naked eye through the aggregation of gold nanoparticles that is triggered by the product of AChE catalyzed acetylthiocholine, with a detection limit of 0.5 ng/mL. Moreover, due to the MCBs have high luminance with perfect uniformity, the detection can also be realized by counting the number of MCBs, with a detection limit of 1 ng/mL. The method is validated with human throat swabs, resulting in a complete consistence with reverse transcription-polymerase chain reaction results. This study reports the first cellular beacons-based method for pathogen detection by the naked eye and broadens the applicability of cell self-synthesized nanoparticles-based immunoassays. Moreover, the MCBs-based method will provide a powerful tool for clinical detection.


Assuntos
Técnicas Biossensoriais , Faringe/microbiologia , Pontos Quânticos/química , Staphylococcus aureus/isolamento & purificação , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Anticorpos/química , Ouro/química , Humanos , Nanoestruturas/química , Reação em Cadeia da Polimerase em Tempo Real , Staphylococcus aureus/genética
5.
Anal Chem ; 89(24): 13105-13111, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29148720

RESUMO

Rapid detection of highly contagious pathogens is the key to increasing the probability of survival and reducing infection rates. We developed a sensitive and quantitative lateral flow assay for detection of Ebola virus (EBOV) glycoprotein with a novel multifunctional nanosphere (RNs@Au) as a reporter. Each RNs@Au contains hundreds of quantum dots and dozens of Au nanoparticles and can achieve enhanced dual-signal readout (fluorescence signal for quantitative detection and colorimetric signal for visual detection). Antibody (Ab) and streptavidin (SA) were simultaneously modified onto the RNs@Au to label the target and act as signal enhancer. After the target was labeled by the Ab-RNs@Au-SA and captured on the test line, biotin-modified RNs@Au was used to amplify the dual signal by the reaction of SA with biotin. The assay enables naked-eye detection of 2 ng/mL glycoprotein within 20 min, and the quantitative detection limit is 0.18 ng/mL. Additionally, the assay has been successfully tested in field work for detecting EBOV in spiked urine, plasma, and tap water samples and is thus a promising candidate for early diagnosis of suspect infections in EBOV-stricken areas.


Assuntos
Ebolavirus/química , Glicoproteínas/análise , Ouro/química , Nanosferas/química , Sistemas Automatizados de Assistência Junto ao Leito , Humanos
6.
Anal Chem ; 87(21): 11105-12, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26461918

RESUMO

Human enterovirus 71 (EV71) is one of the pathogens that causes hand, foot, and mouth disease (HFMD), which generally leads to neurological diseases and fatal complications among children. Since the early clinical symptoms from EV71 infection are very similar to those from Coxsackievirus B3 (CVB3) infection, a robust and sensitive detection method that can be used to distinguish EV71 and CVB3 is urgently needed for prompting medical treatment of related diseases. Herein, based on immunomagnetic nanobeads and fluorescent semiconductor CdSe quantum dots (QDs), a method for simultaneous point-of-care detection of EV71 and CVB3 is proposed. The synchronous detection of EV71 and CVB3 virions was achieved within 45 min with high specificity and repeatability. The limits of detection are 858 copies/500 µL for EV71 and 809 copies/500 µL for CVB3.This proposed method was further validated with 20 human throat swab samples obtained from EV71 or CVB3 positive cases, with results 93.3% consistent with those by the real-time PCR method, demonstrating the potential of this method for clinical quantification of EV71 and CVB3. The method may also facilitate the prevention and treatment of the diseases.


Assuntos
Enterovirus Humano A/isolamento & purificação , Enterovirus Humano B/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Pontos Quânticos , Espectrometria de Fluorescência
7.
Int J Infect Dis ; 131: 32-39, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36967037

RESUMO

OBJECTIVES: From March to June 2021, the reported number of clinically diagnosed endemic typhus in Anhui and Hubei provinces of China nearly increased four-fold compared with the monthly average numbers in last 5 years. An etiological and epidemiological investigation was initiated. METHODS: The clinical specimens from the reported patients and the potential vector ticks were collected for molecular and serological detection, as well as cell culturing assay to identify the potential pathogen. RESULTS: Polymerase chain reaction and sequence analysis of rrs and groEL showed that the pathogen from these patients was Ehrlichia sp., isolated from Haemaphysalis longicornis attached to these patients. The phylogenetic analysis based on 39 Ehrlichia genomes suggested that it should be taxonomically classified as a novel species, tentatively named "Candidatus Ehrlichia erythraense". A total of 19 of 106 cases were confirmed as Candidatus Ehrlichia erythraense infections by polymerase chain reaction, sequencing, and/or serological tests. The most frequent symptoms were fever (100%), rashes (100%), asthenia (100%), anorexia (100%), and myalgia (79%). CONCLUSION: The occurrence of the disease presenting with fever and rashes in Anhui and Hubei provinces was caused by a novel species of the genus Ehrlichia; physicians need to be aware of this newly-discovered pathogen to ensure appropriate testing, treatment, and regional surveillance.


Assuntos
Ehrlichiose , Carrapatos , Animais , Humanos , Ehrlichia/genética , Filogenia , Ehrlichiose/diagnóstico , Ehrlichiose/epidemiologia , China/epidemiologia
8.
Virol Sin ; 37(4): 503-512, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35643410

RESUMO

Group A human rotaviruses (RVAs) annually cause the deaths of 215,000 infants and young children. To understand the epidemiological characteristics and genetic evolution of RVAs, we performed sentinel surveillance on RVA prevalence in a rotavirus-surveillance network in Hubei, China. From 2013 to 2016, a total of 2007 fecal samples from hospital outpatients with acute gastroenteritis were collected from four cities of Hubei Province. Of the 2007 samples, 153 (7.62%) were identified positive for RVA by real-time RT-PCR. RVA infection in Hubei mainly occurred in autumn and winter. The highest detection rate of RVA infection was in 1-2 years old of outpatients (16.97%). No significant difference of RVA positive rate was observed between females and males. We performed a phylogenetic analysis of the G/P genotypes based on the partial VP7/VP4 gene sequences of RVAs. G9P[8] was the most predominant strain in all four years but the prevalence of G2P[4] genotype increased rapidly since 2014. We reconstructed the evolutionary time scale of RVAs in Hubei, and found that the evolutionary rates of the G9, G2, P[8], and P[4] genotypes of RVA were 1.069 â€‹× â€‹10-3, 1.029 â€‹× â€‹10-3, 1.283 â€‹× â€‹10-3 and 1.172 â€‹× â€‹10-3 nucleotide substitutions/site/year, respectively. Importantly, using a molecular clock model, we showed that most G9, G2, P[8], and P[4] genotype strains dated from the recent ancestor in 2005, 2005, 1993, and 2013, respectively. The finding of the distribution of RVAs in infants and young children in Hubei Province will contribute to the understanding of the epidemiological characteristics and genetic evolution of RVAs in China.


Assuntos
Infecções por Rotavirus , Rotavirus , Criança , Pré-Escolar , Diarreia/epidemiologia , Fezes , Feminino , Genótipo , Humanos , Lactente , Masculino , Pacientes Ambulatoriais , Filogenia , Rotavirus/genética , Infecções por Rotavirus/epidemiologia
9.
J Anal Test ; 6(4): 353-364, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35966388

RESUMO

Gold nanoparticles (AuNPs) colorimetric assays based on distance-dependent optical characteristics have been widely employed for bioanalysis. However, this assay is not effective for visually detecting low-concentration targets due to the faint color change. Here, we developed a handheld nano-centrifugal device which could separate the crosslinked and non-crosslinked AuNPs. Results showed that the handheld nano-centrifugal device could easily reach more than 6000 r/min within 10 s simply by stretching and tightening the coiled rope in an appropriate rhythm. Further, combined with the CRISPR/Cas12a nucleic acids recognition system, a field-deployable colorimetric platform termed handheld nano-centrifugal device assisted CRISPR/Cas12a (Hand-CRISPR) has been validated. Moreover, clinical diagnostics applications for Epstein-Barr virus (EBV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection with high sensitivity and accuracy (100% consistency with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) test results) have been demonstrated. Overall, the Hand-CRISPR platform showed great promise in point-of-care-test (POCT) application, expected to become a powerful supplement to the standard nucleic acid testing method in remote or poverty-stricken areas. Supplementary Information: The online version contains supplementary material available at 10.1007/s41664-022-00232-0.

10.
Parasit Vectors ; 13(1): 360, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32690109

RESUMO

BACKGROUND: Schistosoma japonicum is a waterborne parasite that causes schistosomiasis in humans and in more than 40 animal species. Schistosoma japonicum shows distinct genetic differentiation among geographical populations and multiple hosts, but the genetic diversity of different developmental stages of S. japonicum from is less studied. Such studies could elucidate ecological mechanisms in disease transmission by analysing feedbacks in individual physiology and population state. METHODS: After infection using cercariae from a pool of snails shedding together (Method I) and infection using mixed equal numbers of cercariae from individually shed snails (Method II), different developmental stages of S. japonicum were genotyped with microsatellite loci, including 346 cercariae, 701 adult worms and 393 miracidia. Genetic diversity and molecular variation were calculated at different population levels. Kinships (I') among cercariae at intra-snail and inter-snail levels were evaluated. Genetic distance (Dsw) was compared between paired and unpaired worms, and partner changing was investigated through paternity identification for miracidia. RESULTS: The cercaria clones in individual snails varied from 1 to 8 and the kinship of cercariae within individual snails was significant higher (P < 0.001) than that among different snails after deleting near-identical multi-locus genotypes (niMLGs). The allelic diversity of worms in Method I was lower (P < 0.001) than that in Method II, and allele frequency among mice in Method I was also less consistent. The parents of some miracidia were worms that were not paired when collected. The Dsw between each female of paired and unpaired males was much larger (P < 0.001) than that between the female and male in each pair. CONCLUSIONS: Most of the infected snails contained multiple miracidia clones. The aggregation of genetically similar S. japonicum miracidia in individual snails and the unbalanced distribution of miracidia among snails suggests a non-uniform genetic distribution of cercariae among snails in the field. This further influenced the genetic structure of adult worms from infections with different cercariae sampling methods. Schistosoma japonicum in mice can change paired partner, preferring to mate with genetically similar worms. These characteristics provide implications for understanding the balance in genetic diversity of S. japonicum related to the transmission of schistosomiasis.


Assuntos
Schistosoma japonicum/genética , Esquistossomose Japônica/transmissão , Caramujos/parasitologia , Animais , Cercárias/genética , Variação Genética , Técnicas de Genotipagem , Estágios do Ciclo de Vida/genética , Preferência de Acasalamento Animal , Camundongos , Repetições de Microssatélites/genética
11.
Chin Med J (Engl) ; 133(9): 1015-1024, 2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32004165

RESUMO

BACKGROUND: Human infections with zoonotic coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, have raised great public health concern globally. Here, we report a novel bat-origin CoV causing severe and fatal pneumonia in humans. METHODS: We collected clinical data and bronchoalveolar lavage (BAL) specimens from five patients with severe pneumonia from Wuhan Jinyintan Hospital, Hubei province, China. Nucleic acids of the BAL were extracted and subjected to next-generation sequencing. Virus isolation was carried out, and maximum-likelihood phylogenetic trees were constructed. RESULTS: Five patients hospitalized from December 18 to December 29, 2019 presented with fever, cough, and dyspnea accompanied by complications of acute respiratory distress syndrome. Chest radiography revealed diffuse opacities and consolidation. One of these patients died. Sequence results revealed the presence of a previously unknown ß-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. These isolates showed 79.0% nucleotide identity with the sequence of SARS-CoV (GenBank NC_004718) and 51.8% identity with the sequence of MERS-CoV (GenBank NC_019843). The virus is phylogenetically closest to a bat SARS-like CoV (SL-ZC45, GenBank MG772933) with 87.6% to 87.7% nucleotide identity, but is in a separate clade. Moreover, these viruses have a single intact open reading frame gene 8, as a further indicator of bat-origin CoVs. However, the amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating that these viruses might use the same receptor. CONCLUSION: A novel bat-borne CoV was identified that is associated with severe and fatal respiratory disease in humans.


Assuntos
Betacoronavirus , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Adulto , Idoso , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , COVID-19 , Infecções por Coronavirus/diagnóstico por imagem , Infecções por Coronavirus/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/diagnóstico por imagem , Pneumonia Viral/terapia , SARS-CoV-2 , Tomografia por Raios X , Resultado do Tratamento
13.
Biosens Bioelectron ; 99: 186-192, 2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-28756324

RESUMO

Point-of-care detection of human enterovirus 71 (EV71), the major pathogen that causes hand, foot, and mouth disease (HFMD) among children, is urgently needed for early diagnosis and control of related epidemics. A colorimetric and electrochemical immunosensor for point-of-care detection of EV71 has been developed based on dual-labeled magnetic nanobeads amplification. The dual-labeled magnetic nanobeads (DL-MBs) are fabricated by simultaneous immobilization of EV71 monoclonal antibody (mAb) and horseradish peroxidase (HRP) on magnetic nanobeads. By capturing EV71 virions in 20µL sample on mAb modified AuNPs-coated ITO electrode and subsequently binding with DL-MBs, with the addition of TMB and H2O2, colorimetric signals corresponding to EV71 with a concentration of 1.0ngmL-1 can be directly read out by naked eyes, making it possible towards point-of-care detection of the virus. Furthermore, on the reduction of oxidized TMB on the electrode, electrochemical signal can be detected in the same detection cell without solution transfer, with a detection limit of 0.01ngmL-1. Validated with clinical samples, the colorimetric and electrochemical immunosensor shows a complete consistency with reverse transcription-polymerase chain reaction (RT-PCR) results. So far as we know, this is the first report on EV71 detection using electrochemical method. The merits of this assay, including high sensitivity, ability for colorimetric detection and easy to operation, ensure a promising future in point-of-care diagnostics of virus related diseases.


Assuntos
Técnicas Biossensoriais , Colorimetria , Técnicas Eletroquímicas , Enterovirus Humano A/isolamento & purificação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Enterovirus Humano A/patogenicidade , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao Leito
14.
Eur J Hum Genet ; 15(12): 1246-51, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17625508

RESUMO

The ventricular septal defect (VSD) is the most common congenital heart defect and no candidate susceptibility gene has been identified. Endocardial cushion and outflow septal morphogenesis, malalignment of which induces VSD, have been suggested to be mediated by the vascular endothelial growth factor (VEGF). Three single-nucleotide polymorphism (SNP) variants in promoter and 5'-UTR region of the VEGF gene, C-2578A (rs699947), G-1154A (rs1570360) and G-634C (rs2010963), were reported to alter its expression. We assessed the association in a Chinese population between these SNPs and VSD using a double approach: case-control and TDT designs. Among the three SNPs, only -634C allele was less frequently present in 222 patients compared to 352 controls (odds ratio: 0.76, 95% CI: 0.59-0.97, X(2)=5.06, P=0.024, not significant after a Bonferroni correction). This was significantly less transmitted to VSD patients (trios: 142) (odds ratio: 0.39, 95% CI: 0.25-0.62, X(2)=8.11, df=1, P=0.004, corrected P=0.024). A similar result was observed for haplotype -2578C/-1154G/-634C allele in both studies (in TDT: X(2)=7.51, df=1, P=0.006, corrected P=0.048). All these associations for the first time demonstrated that -634C allele was in a significant protective association against VSD, suggesting that VEGF dysregulation was involved in the pathological processes of VSD.


Assuntos
Citosina , Guanina , Comunicação Interventricular/genética , Comunicação Interventricular/prevenção & controle , Desequilíbrio de Ligação/genética , Polimorfismo de Nucleotídeo Único/genética , Fator A de Crescimento do Endotélio Vascular/genética , Adolescente , Alelos , Estudos de Casos e Controles , Criança , Pré-Escolar , Família , Feminino , Haplótipos , Humanos , Lactente , Masculino , Fator A de Crescimento do Endotélio Vascular/sangue
15.
ACS Appl Mater Interfaces ; 9(11): 9416-9425, 2017 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-28241111

RESUMO

Enrichment and purification of bacteria from complex matrices are crucial for their detection and investigation, in which magnetic separation techniques have recently show great application advantages. However, currently used magnetic particles all have their own limitations: Magnetic microparticles exhibit poor binding capacity with targets, while magnetic nanoparticles suffer slow magnetic response and high loss rate during treatment process. Herein, we used a highly controllable layer-by-layer assembly method to fabricate quick-response magnetic nanospheres (MNs), and with Salmonella typhimurium as a model, we successfully achieve their rapid and efficient enrichment. The MNs combined the advantages of magnetic microparticles and nanoparticles. On the one hand, the MNs had a fast magnetic response, and almost 100% of the MNs could be recovered by 1 min attraction with a simple magnetic scaffold. Hence, using antibody conjugated MNs (immunomagnetic nanospheres, IMNs) to capture bacteria hardly generated loss and did not need complex separation tools or techniques. On the other hand, the IMNs showed much excellent capture capacity. With 20 min interaction, almost all of the target bacteria could be captured, and even only one bacterium existing in the samples was not missed, comparing with the immunomagnetic microparticles which could only capture less than 50% of the bacteria. Besides, the IMNs could achieve the same efficient enrichment in complex matrices, such as milk, fetal bovine serum, and urine, demonstrating their good stability, strong anti-interference ability, and low nonspecific adsorption. In addition, the isolated bacteria could be directly used for culture, polymerase chain reaction (PCR) analyses, and fluorescence immunoassay without a release process, which suggested our IMNs-based enrichment strategy could be conveniently coupled with the downstream identification and analysis techniques. Thus, the MNs provided by this work showed great superiority in bacteria enrichment, which would be a promising tool for bacteria detection and investigation.


Assuntos
Nanosferas , Animais , Separação Imunomagnética , Leite , Salmonella typhimurium
16.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 23(3): 250-5, 2006 Jun.
Artigo em Zh | MEDLINE | ID: mdl-16767657

RESUMO

OBJECTIVE: To ascertain 5 short tandem repeat (STR) markers as qualified tools for detecting chromosome 22q11 deletion and to understand the prevalence and clinical importance of the deletions in patients with congenital heart diseases (CHD) from Chinese Han population. METHODS: The authors selected 5 new tetranucleotide repeat markers, 22D_4_1, 22D_4_2, 22D_4_3, 22D_4_4 and D22S873 located in the proximal region of chromosome 22q11 deletion. One hundred and sixty-three unselected CHD patients and their unaffected parents were analyzed by genotyping of these new tetranucleotide STR markers to detect 22q11 deletion. With fluorescence in situ hybridization (FISH, LSI dual color DNA probe), the deletion status was confirmed in all patients with deletions and some patients without deletions. RESULTS: The heterozygosity of these STR markers in normal population was more than 0.7, except for 22D_4_1 and 22D_4_2 that were 0.65 and 0.52 respectively. Twelve cases of 163 CHD patients (7.36%) had the deletions at chromosome 22q11. The deletions were confirmed in 9 of 12 patients by FISH, except for 2 cases who had unique nested deletion and 1 case who had nested distal deletion. One hundred and ten patients were associated with ventricular septal defect (VSD); and 9 (8.18%) of these cases had microdeletion. Twenty-one patients were associated with tetralogy of Fallot (TOF); and 3 (14.3%) of these cases had microdeletion. CONCLUSION: This study demonstrated that genotyping of 5 STR markers was a useful mean of detecting 22q11 microdeletion in clinical diagnosis owing to its rapid experimental procedure, cost effectiveness and high resolution. 22q11 deletion was common in CHD patients, particularly in VSD and TOF patients, from Chinese Han population.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 22/genética , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/diagnóstico , Humanos , Hibridização in Situ Fluorescente , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase
17.
Virol Sin ; 26(6): 418-27, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22160942

RESUMO

A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang, Hubei and Guangdong between June and November 2009, were provided by local CDC laboratories. After MDCK cell culture, 57 Influenza A Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing. A total of 39 HA sequences, 52 NA sequences, 36 PB2 sequences, 31 PB1 sequences, 40 PA sequences, 48 NP sequences, 51 MP sequences and 36 NS sequences were obtained, including 20 whole genome sequences. Sequence comparison revealed they shared a high degree of homology (96%-99%) with known epidemic strains (A/California/04/2009(H1N1). Phylogenetic analysis showed that although the sequences were highly conserved, they clustered into a small number of groups with only a few distinct strains. Site analysis revealed three substitutions at loop 220 (221-228) of the HA receptor binding site in the 39 HA sequences: A/Hubei/86/2009 PKVRDQEG → PKVRDQEA, A/Zhejiang/08/2009 PKVRDQEG → PKVRDQER, A/Hubei/75/2009 PKVRDQEG → PKVRDQGG, the A/Hubei/75/2009 was isolated from an acute case, while the other two were from patients with mild symptoms. Other key sites such as 119, 274, 292 and 294 amino acids of NA protein, 627 of PB2 protein were conserved. Meanwhile, all the M2 protein sequences possessed the Ser32Asn mutation, suggesting that these viruses were resistant to adamantanes. Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/epidemiologia , Influenza Humana/virologia , Pandemias , Sequência de Aminoácidos , China/epidemiologia , Variação Genética , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/classificação , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética
19.
Mol Cell Probes ; 20(6): 359-65, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16774813

RESUMO

The 22q11.2 deletion syndrome is the most common microdeletion syndrome mainly characterized by hemizygous deletions and congenital heart defect (CHD). By using polymerase chain reaction (PCR), genotyping of short tandem repeat polymorphic (STRP) markers is a common and powerful means of detecting microdeletion. We have developed five new tetranucleotide repeat markers, 22D_4_1, 22D_4_2, 22D_4_3, 22D_4_4 and D22S873 in the proximal region of 22q11.2 deletion. To ascertain whether these markers could be used reliably in genotyping, we performed genotyping analysis on 200 unrelated individuals from a Chinese Han population and 67 CHD patients and their unaffected parents. Population data showed that the five markers met Hardy-Weinberg expectations and were highly polymorphic. By using the five markers, six of 67 CHD patients were determined to have a deletion within chromosome 22q11.2. Compared with dinucleotide markers, tetranucleotide markers produce weaker stutter bands and have no artificial multiband patterns. PCR amplification results from the five new tetranucleotide STRP markers were unambiguous and easier to interpret in genotyping. This study demonstrated that the five markers were efficient and reliable suggesting that genotyping using tetranucleotide STRP markers is an alterative approach to detect the deletion on chromosome 22q11.2 in clinical diagnosis and for genetic consultation.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 22 , Marcadores Genéticos , Repetições de Microssatélites , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Frequência do Gene , Genótipo , Haplótipos , Heterozigoto , Humanos , Lactente , Masculino , Dados de Sequência Molecular
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