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1.
J Virol ; 97(12): e0123223, 2023 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-38051045

RESUMEN

IMPORTANCE: Over the past decade, increasing evidence has shown that circular RNAs (circRNAs) play important regulatory roles in viral infection and host antiviral responses. However, reports on the role of circRNAs in Zika virus (ZIKV) infection are limited. In this study, we identified 45 differentially expressed circRNAs in ZIKV-infected A549 cells by RNA sequencing. We clarified that a downregulated circRNA, hsa_circ_0007321, regulates ZIKV replication through targeting of miR-492 and the downstream gene NFKBID. NFKBID is a negative regulator of nuclear factor-κB (NF-κB), and we found that inhibition of the NF-κB pathway promotes ZIKV replication. Therefore, this finding that hsa_circ_0007321 exerts its regulatory role on ZIKV replication through the miR-492/NFKBID/NF-κB signaling pathway has implications for the development of strategies to suppress ZIKV and possibly other viral infections.


Asunto(s)
ARN Circular , Infección por el Virus Zika , Virus Zika , Humanos , MicroARNs/genética , MicroARNs/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Circular/genética , Transducción de Señal , Virus Zika/genética , Virus Zika/metabolismo , Infección por el Virus Zika/genética
2.
J Med Virol ; 96(3): e29533, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38483048

RESUMEN

Cytidine/uridine monophosphate kinase 2 (UMP-CMP kinase 2, CMPK2) has been reported as an antiviral interferon-stimulated gene (ISG). We previously observed that the expression of CMPK2 was significantly upregulated after Zika Virus (ZIKV) infection in A549 cells. However, the association and the underlying mechanisms between CMPK2 induction and ZIKV replication remain to be determined. We investigated the induction of CMPK2 during ZIKV infection and the effect of CMPK2 on ZIKV replication in A549, U251, Vero, IFNAR-deficient U5A and its parental 2fTGH cells, Huh7 and its RIG-I-deficient derivatives Huh7.5.1 cells. The activation status of Jak-STAT signaling pathway was determined by detecting the phosphorylation level of STAT1, the activity of interferon stimulated response element (ISRE) and the expression of several interferon stimulated genes (ISGs). We found that ZIKV infection induced CMPK2 expression through an IFNAR and RIG-I dependent manner. Overexpression of CMPK2 inhibited while CMPK2 knockdown promoted ZIKV replication in A549 and U251 cells. Mechanically, we found that CMPK2 overexpression increased IFNß expression and activated Jak/STAT signaling pathway as shown by the increased level of p-STAT1, enhanced activity of ISRE, and the upregulated expression of downstream ISGs. These findings suggest that ZIKV infection induced CMPK2 expression, which inhibited ZIKV replication and serves as a positive feedback regulator for IFN-Jak/STAT pathway.


Asunto(s)
Interferón Tipo I , Nucleósido-Fosfato Quinasa , Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/metabolismo , Transducción de Señal , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Factores de Transcripción STAT/farmacología , Interferón Tipo I/genética , Replicación Viral , Receptores Inmunológicos
3.
J Med Virol ; 96(4): e29624, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38647075

RESUMEN

Respiratory infections pose a serious threat to global public health, underscoring the urgent need for rapid, accurate, and large-scale diagnostic tools. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, combined with isothermal amplification methods, has seen widespread application in nucleic acid testing (NAT). However, achieving a single-tube reaction system containing all necessary components is challenging due to the competitive effects between recombinase polymerase amplification (RPA) and CRISPR/Cas reagents. Furthermore, to enable precision medicine, distinguishing between bacterial and viral infections is essential. Here, we have developed a novel NAT method, termed one-pot-RPA-CRISPR/Cas12a, which combines RPA with CRISPR molecular diagnostic technology, enabling simultaneous detection of 12 common respiratory pathogens, including six bacteria and six viruses. RPA and CRISPR/Cas12a reactions are separated by paraffin, providing an independent platform for RPA reactions to generate sufficient target products before being mixed with the CRISPR/Cas12a system. Results can be visually observed under LED blue light. The sensitivity of the one-pot-RPA-CRISPR/Cas12a method is 2.5 × 100 copies/µL plasmids, with no cross-reaction with other bacteria or viruses. Additionally, the clinical utility was evaluated by testing clinical isolates of bacteria and virus throat swab samples, demonstrating favorable performance. Thus, our one-pot-RPA-CRISPR/Cas12a method shows immense potential for accurate and large-scale detection of 12 common respiratory pathogens in point-of-care testing.


Asunto(s)
Bacterias , Sistemas CRISPR-Cas , Técnicas de Diagnóstico Molecular , Infecciones del Sistema Respiratorio , Virus , Bacterias/genética , Bacterias/aislamiento & purificación , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/microbiología , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Recombinasas/genética , Recombinasas/metabolismo , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/virología , Infecciones del Sistema Respiratorio/microbiología , Sensibilidad y Especificidad , Virosis/diagnóstico , Virus/genética , Virus/aislamiento & purificación
4.
Mikrochim Acta ; 191(5): 271, 2024 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-38632191

RESUMEN

Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5 CFU/mL in a time of 70 min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.


Asunto(s)
Anticuerpos , Shigella flexneri , Humanos , Luz Azul , Fluorescencia , Recombinasas
5.
J Med Virol ; 95(8): e29006, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37548473

RESUMEN

A small percentage of couples who regularly donated blood in China tested positive for HBsAg. Although it is well known that blood donors can acquire hepatitis B virus (HBV) infection from a chronically infected sexual partner, the prevalence of occult hepatitis B infections (OBIs) among blood donations from partners of HBV-infected chronically infected spouses and the risk to blood safety remain poorly understood. Among 212 763 blood donors, 54 pairs of couples (108 donations) were enrolled because one partner tested positive for HBsAg. Several molecular and serological examinations were conducted. The origin of HBV transmission between sexual partners was investigated further. Also evaluated was the potential risk of HBV infection with OBIs. We identified 10 (10/54, 18.6%) sexual partners of chronically infected HBV donors who were positive for HBV DNA, including five samples (9.3%) with OBIs, of which 3 (3/54, 5.6%, 1 in 70 921 donations) passed the routine blood screening tests. Seven of the 10 HBV-DNA-positive couples contracted the virus possibly through sexual or close contact. Among infected couples, immune escape mutations were observed. A high prevalence of OBIs was found among the partners of chronically infected HBV blood donors, posing a potential threat to blood safety.


Asunto(s)
Donantes de Sangre , Seguridad de la Sangre , Hepatitis B , Esposos , Seguridad de la Sangre/estadística & datos numéricos , Hepatitis B/epidemiología , Hepatitis B/prevención & control , Hepatitis B/transmisión , Esposos/estadística & datos numéricos , Prevalencia , China/epidemiología , Donantes de Sangre/estadística & datos numéricos , Virus de la Hepatitis B , Humanos , Masculino , Femenino , Adulto Joven , Adulto , Persona de Mediana Edad
6.
Acta Haematol ; 145(4): 347-353, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35134796

RESUMEN

Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it has spread rapidly around the world and caused a serious global social crisis. During the epidemic, the blood collection and supply industry have been greatly impacted, due to the sharply dropped blood donors and transfusion transmission risk of SARS-CoV-2. Many infected individuals are asymptomatic and they may donate blood without awareness of the infection or before symptoms appear. In addition, viral RNAs have been detected in the blood of some patients infected with SARS-CoV-2. Although no infectious SARS-CoV-2 virus was found in the blood nor the blood components, there is a risk of transmission through blood transfusion which may endanger blood safety, especially during the pandemic period. This review briefly introduces the biological characteristics, epidemiology of SARS-CoV-2, with a particular focus on SARS-CoV-2 infection and blood safety.


Asunto(s)
COVID-19 , Seguridad de la Sangre , COVID-19/epidemiología , Humanos , Pandemias , ARN Viral , SARS-CoV-2
7.
J Dairy Sci ; 105(4): 2849-2857, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35151480

RESUMEN

In recent years, food safety incidents caused by Escherichia coli have occurred and have endangered human health. Due to the complex matrix of milk samples and the long pretreatment time, the existing methods cannot quickly detect E. coli in milk samples. It is necessary to enrich the E. coli in the complex matrix to improve the detection sensitivity. The E. coli outer membrane protein A (OmpA) is widely present on the cell membrane of E. coli and may be used as a new target to enrich E. coli. In this study, the purified recombinant OmpA protein was used to immunize BALB/c mice to produce polyclonal antibody. Immunomagnetic beads were combined with the polyclonal antibody to enrich the E. coli in the artificially contaminated milk samples. The products of immunoprecipitation were further used for PCR assay. The bacteria in the PCR sample can be pre-enriched, and the limit of detection is 10 × 100 cfu/mL, which is about 100 times more sensitive than samples not processed by this method. Then, the artificially contaminated milk, coffee, juice, and soybean milk samples were tested separately, and it was found that the E. coli gene could be amplified. The whole analysis time was about 120 min, including the enrichment of bacteria and the detection of eluate. We found that OmpA combined with immunomagnetic beads was more efficient, fast, and convenient than the conventional method. Bacteria can be enriched more efficiently without extracting genomic DNA and culturing bacteria. Therefore, this method has potential value for improving the detection sensitivity and shortening the detection time of E. coli in food samples.


Asunto(s)
Escherichia coli O157 , Animales , Proteínas de la Membrana Bacteriana Externa , Escherichia coli O157/genética , Microbiología de Alimentos , Separación Inmunomagnética/métodos , Separación Inmunomagnética/veterinaria , Ratones , Leche/microbiología
8.
Foodborne Pathog Dis ; 18(9): 640-646, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34292761

RESUMEN

Escherichia coli, Staphylococcus aureus, Shigella, Pseudomonas aeruginosa, and Klebsiella pneumoniae are common foodborne pathogens. In this study, the light-induced PMAxx-coupled multiplex PCR (PMAxx-mPCR) was established to detect the aforementioned five foodborne pathogens in fresh juice at the same time. Moreover, PMAxx pretreatment could effectively distinguish live bacteria from dead bacteria. The optimized PMAxx pretreatment conditions were incubation with a final concentration of 10 µmol/L PMAxx for 10 min and then photolysis for 8 min. After PMAxx pretreatment, the difference in Ct values with or without PMAxx was determined by quantitative real-time PCR. The results showed a significant difference in Ct value before and after PMAxx treatment. Finally, the bacteria-contaminated fresh juice samples treated with PMAxx dye were detected by mPCR. The detection limit of PMAxx-mPCR was 102 colony-forming units (CFU)/mL for E. coli, Shigella, P. aeruginosa, and K. pneumoniae and 103 CFU/mL for S. aureus. Compared with mPCR detection of samples without PMAxx treatment, the proposed method solved the false-positive problem due to dead bacteria. Hence, an accurate and efficient method for the simultaneous detection of five types of pathogenic bacteria was established. This method could be applied to analytical procedures for ensuring food safety.


Asunto(s)
Reacción en Cadena de la Polimerasa Multiplex , Staphylococcus aureus , Bacterias/genética , Escherichia coli , Microbiología de Alimentos , Reacción en Cadena en Tiempo Real de la Polimerasa , Staphylococcus aureus/genética
9.
Clin Lab ; 65(4)2019 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-30969069

RESUMEN

BACKGROUND: Escherichia coli is the most common pathogenic bacteria that frequently causes life-threatening opportunistic human infections, diarrhea, and septicemia in immunocompromised hosts. METHODS: This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of a hypothetical protein from an E. coli-specific gene (GenBank ID: 13702648). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65ºC for 30 minutes and 80ºC for 2 minutes, whereas the reaction system contained 5.2 mM Mg2+, 8 U of Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide, and 0.2 and 1.6 µM of the outer and inner primers, respectively. The LAMP method was evaluated using 240 strains of E. coli and 150 strains of non-E. coli. RESULTS: Positive reactions were observed on all 240 strains of E. coli while all non-E. coli strains were negative. Plasmids with the specific gene and mice blood with E. coli were used for sensitivity analysis. The detection limit of LAMP was 100 bacterium/reaction. CONCLUSIONS: Results showed that the LAMP targeted to the hypothetical protein (GenBank ID: 13702648) is a fast, specific, sensitive, inexpensive, and suitable method for the detection of E. coli.


Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Escherichia coli/genética , Técnicas de Amplificación de Ácido Nucleico , Animales , Cartilla de ADN/genética , Diarrea/microbiología , Humanos , Huésped Inmunocomprometido , Límite de Detección , Ratones , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
10.
CRISPR J ; 7(3): 156-167, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38922054

RESUMEN

CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing.


Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Edición Génica , Genes Reporteros , Luciferasas , Plásmidos , ARN Guía de Sistemas CRISPR-Cas , Edición Génica/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Plásmidos/genética , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Virus de la Hepatitis B/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo
11.
Food Chem ; 374: 131828, 2022 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-34915371

RESUMEN

A quantitative comparison of the proteomes during different periods of the formation of egg yolk, from yellow follicles (YF), small hierarchical follicles (9-12 mm, SF), and the largest hierarchical follicle (LF), was performed. The abundance of major egg yolk proteins (apolipoprotein B and vitellogenins) changed significantly during the yolk formation, and several protein hydrolases and isomerases, which may be related to the processing of egg yolk proteins, also varied significantly. The binding proteins for three vitamins (retinol, riboflavin, and vitamin D) and cholesterol were all most abundant in the LF period, suggesting that these nutrients were transported mainly at the final period of the egg yolk formation. Immunoglobulins decreased and complement proteins increased as formation progressed, indicating the dynamic nature of the egg yolk immune system. These results are helpful for understanding the nutrient composition, the formation of assembly structure, the preservation and processing properties of egg yolk.


Asunto(s)
Yema de Huevo , Proteómica , Animales , Pollos , Proteínas del Huevo , Proteoma
12.
Front Bioeng Biotechnol ; 10: 845688, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35265606

RESUMEN

Shigella flexneri is a serious threat to global public health, and a rapid detection method is urgently needed. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system is widely used in gene editing, gene therapy, and in vitro diagnosis. Here, we combined loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a to develop a novel diagnostic test (CRISPR/Cas12a-E-LAMP) for the diagnosis of S. flexneri. The CRISPR/Cas12a-E-LAMP protocol conducts LAMP reaction for S. flexneri templates followed by CRISPR/Cas12a detection of predefined target sequences. LAMP primers and sgRNAs were designed to the highly conserved gene hypothetical protein (accession: AE014073, region: 4170556-4171,068) of S. flexneri. After the LAMP reaction at 60°C for 20 min, the pre-loaded CRISPR/Cas12a regents were mixed with the LAMP products in one tube at 37°C for 20 min, and the final results can be viewed by naked eyes with a total time of 40 min. The sensitivity of CRISPR/Cas12a-E-LAMP to detect S. flexneri was 4 × 100 copies/µl plasmids and without cross-reaction with other six closely related non-S. flexneri. Therefore, the CRISPR/Cas12a-E-LAMP assay is a useful method for the reliable and quick diagnosis of S. flexneri and may be applied in other pathogen infection detection.

13.
Int J Infect Dis ; 112: 55-62, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34517048

RESUMEN

OBJECTIVES: Shigella flexneri (S. flexneri) is prevalent worldwide and the most common Shigella in many countries, causing highly contagious diarrhea, which seriously threatens public health. This study aimed to develop a colorimetric loop-mediated isothermal amplification (LAMP) for the rapid, accurate, and visualization detection of S. flexneri. METHODS: According to the screened specific genes of S. flexneri, three groups of LAMP primers were designed and evaluated, and the colorimetric LAMP reaction volume was optimized. The specificity of the colorimetric LAMP was validated by 20 S. flexneri and 96 non-S. flexneri clinical isolates. In addition, the sensitivity of the developed assay was evaluated by the serial 10-fold dilutions of plasmid DNA. RESULTS: A colorimetric LAMP assay was developed based on the specific S. flexneri hypothetical protein gene (Accession: AE014073 Region: 4170556.4171068). The colorimetric LAMP method had good specificity for detecting S. flexneri and enabled detection of S. flexneri within 30 minutes, with a plasmid detection limit of 7*10° copies/µL. The results of amplification could be easily identified by color. CONCLUSIONS: This colorimetric LAMP assay could be used for rapid and accurate diagnosis of S. flexneri infection, especially in remote hospitals and laboratories with under-equipped medical facilities, and in situations where an urgent diagnosis is needed.


Asunto(s)
Colorimetría , Shigella flexneri , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , Shigella flexneri/genética
14.
PeerJ ; 8: e8944, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32309051

RESUMEN

BACKGROUND: Antibiotics are highly effective drugs used in the treatment of infectious diseases. Aminoglycoside antibiotics are one of the most common antibiotics in the treatment of bacterial infections. However, the development of drug resistance against those medicines is becoming a serious concern. AIM: This study aimed to develop an efficient, rapid, accurate, and sensitive detection method that is applicable for routine clinical use. METHODS: Escherichia coli was used as a model organism to develop a rapid, accurate, and reliable multiplex polymerase chain reaction (M-PCR) for the detection of four aminoglycoside modifying enzyme (AME) resistance genes Aac(6')-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3')-Ia. M-PCR was used to detect the distribution of AME resistance genes in 237 clinical strains of E. coli. The results were verified by simplex polymerase chain reaction (S-PCR). RESULTS: Results of M-PCR and S-PCR showed that the detection rates of Aac(6')-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3')-Ia were 32.7%, 59.2%, 23.5%, and 16.8%, respectively, in 237 clinical strains of E. coli. Compared with the traditional methods for detection and identification, the rapid and accurate M-PCR detection method was established to detect AME drug resistance genes. This technique can be used for the clinical detection as well as the surveillance and monitoring of the spread of those specific antibiotic resistance genes.

15.
ACS Omega ; 5(16): 9301-9310, 2020 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-32363280

RESUMEN

Antimicrobial peptides (AMPs) are a class of templates with application potential for drug development. Amphibians are important sources of AMPs. Duttaphrynus melanostictus is the main source of traditional Chinese medicine "Chansu", which has anti-infection effect while without a clear mechanism. This study aimed to find the cathelicidin peptide in D. melanostictus and then investigate the activity in vivo and in vitro, and an AMP-encoding gene (cathelicidin-DM, GenBank: KJ820824.1) was obtained from the constructed cDNA library of D. melanostictus. The MIC test and SYTOX Green uptake were used for the evaluation of the bactericidal capacity and mechanisms. The serum stability tests were used for the evaluation of the application potential. The skin wound infection model and in vivo imaging were used for in vitro application of possibility evaluation. The results showed that cathelicidin-DM was a 37 amino acid AMP with good bactericidal ability, which was similar to melittin: both can kill bacteria within 15 min. Moreover, cathelicidin-DM exhibits good therapeutic potential in the mouse wound infection model, and it can be enriched to the site of infection for treatment. Thus, cathelicidin-DM could be a new template for antimicrobial drug development given its good antibacterial activity in vivo and in vitro.

16.
J Biomed Nanotechnol ; 15(5): 1061-1071, 2019 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-30890236

RESUMEN

Klebsiella pneumoniae is a common human pathogen that causes several nosocomial chronic infections. Many methods have been reported to detect Klebsiella pneumonia; however, a simple, rapid, and visual method remains to be developed. Here, we developed a rapid, visual detection method employing a nanoparticle immunosensor (NPIS) with a sensitivity of 8 CFU mL-1. Two monoclonal antibodies (mAbs) recognizing different antigenic determinants on the surface of Klebsiella pneumonia were screened by an indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, and mass spectrometry assay. The mAb 1E6 was conjugated to magnetic nanoparticles (MNPs) to generate the immuno-magnetic nanoparticles (IMNPs), whereas the horseradish peroxidase (HRP)-mAb probe was synthesized by conjugating the mAb 2F1 with HRP. Thereafter, the NPIS was developed based on the IMNPs and the HRP-mAb probe, and the total assay time for the detection of Klebsiella pneumoniae was ∼60 min. A rapid, sensitive method was developed for screening Klebsiella pneumoniae in clinical samples. The NPIS developed in this study can detect Klebsiella pneumoniae without the enrichment of bacteria and the extraction of the genome; therefore, it can be used as a primary screening method to compliment other methods in the detection of Klebsiella pneumoniae.


Asunto(s)
Klebsiella pneumoniae , Nanopartículas de Magnetita , Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática , Peroxidasa de Rábano Silvestre , Humanos
17.
Exp Ther Med ; 17(1): 131-136, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30651773

RESUMEN

Pseudomonas aeruginosa is one of the three most pathogenic bacteria that frequently cause life-threatening opportunistic human infections, pneumonia, and lower respiratory tract infections in immunocompromised hosts, particularly in the burns ward. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of P. aeruginosa-specific gene hypothetical protein (GenBank ID: 882161). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65°C for 30 min and 80°C for 2 min, whereas the reaction system contained 5.2 mM Mg2+, 8 U Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide and 0.2 and 1.6 µM of the outer and inner primers, respectively. The LAMP method was evaluated using 150 P. aeruginosa and 170 non-P. aeruginosa strains. Positive reactions were observed on 150 P. aeruginosa strains, whereas all non-P. aeruginosa strains exhibited negative results. Plasmids with the specific gene and mouse blood with P. aeruginosa were used for sensitivity assay. The detection limit of LAMP was 1 bacterium/reaction. Results indicated that the LAMP method targeted to hypothetical protein is a fast, specific, sensitive, inexpensive and suitable method for detection of P. aeruginosa.

18.
Braz J Med Biol Res ; 52(3): e8186, 2019 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-30916220

RESUMEN

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.


Asunto(s)
Genes Bacterianos , Klebsiella pneumoniae/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Cartilla de ADN/genética , Cartilla de ADN/aislamiento & purificación , Klebsiella pneumoniae/aislamiento & purificación , Límite de Detección , Plásmidos/genética , Plásmidos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Temperatura , Factores de Tiempo
19.
Braz. j. med. biol. res ; 52(3): e8186, 2019. tab, graf
Artículo en Inglés | LILACS | ID: biblio-989465

RESUMEN

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.


Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , Genes Bacterianos , Klebsiella pneumoniae/genética , Plásmidos/aislamiento & purificación , Plásmidos/genética , Temperatura , Factores de Tiempo , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Cartilla de ADN/aislamiento & purificación , Cartilla de ADN/genética , Límite de Detección , Klebsiella pneumoniae/aislamiento & purificación
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