ABSTRACT
Some serotypes of enterovirus (EV) may lead to transient and symptomatic gastrointestinal infections while others are commensal residents of the human gut. To determine whether certain EV types are more often associated with diarrhea, we conducted a preliminary study on the prevalence of EV serotypes and common diarrhea viruses in fecal samples of diarrhea children and healthy controls. EV was tested with one step nest polymerase chain reaction and typed by direct sequencing while common causative diarrhea viruses rotavirus (RV), norovirus (NoV), adenovirus (AdV), bocavirus (HBoV), and astrovirus (AstV) were screened with multiplex PCR assays. Human Rhinovirus (HRV) and human EVs that were present in both groups were further quantified and their odds ratios (OR) were calculated. Enteric pathogens were detected in 89 (32.6%) of 273 children with diarrhea and included human EVs (51, 18.68%), HRV (32, 11.72%), RV (38, 13.92%), AdV (24, 8.79%), NoVGII (16, 8.79%), HBoV (8, 2.93%) and AstV (3, 1.09%). Potential enteric pathogens were found in 25 (6.93%) of 361 healthy controls and included human EV (59, 16.34%), HRV (8, 2.22%), RV (1, 0.28%), NoVGII (5, 1.39%), AstV (2, 0.55%), AdV (16, 4.43%) and HBoV (1, 0.28%). In addition, EV71, echovirus 3,9,14,25 and coxsackievirus A14 existed in healthy controls only, while HRV, echovirus11,18, coxsackievirus A2,4,6 and B2,4 were found in both patients and healthy controls. OR assessment confirmed a strong association of HRV (P < 0.001) and a weak one for echovirus 11 and coxsackievirus A6 with diarrhea (P > 0.05). Our results indicate the diversity of EV serotypes in diarrhea and healthy control groups varies, and the potential etiological role of HRV in diarrhea.
Subject(s)
Diarrhea/virology , Enterovirus Infections/virology , Enterovirus/isolation & purification , Case-Control Studies , Child, Preschool , Feces/virology , Female , Humans , Infant , Infant, Newborn , MaleABSTRACT
BACKGROUND: Increasing number of hospitalized children with community acquired pneumonia (CAP) is co-detected with Mycoplasma pneumoniae (Mp). The clinical characteristics and impact of Mp co-detected with other bacterial and/or viral pathogens remain poorly understood. The purpose of this study was to evaluate the demographic and clinical features of CAP children with Mp mono-detection and Mp co-detection. METHODS: A total of 4148 hospitalized children with CAP were recruited from January to December 2017 at the Children's Hospital of Hebei Province, affiliated to Hebei Medical University. A variety of respiratory viruses, bacteria and Mp were detected using multiple modalities. The demographic and clinical features of CAP children with Mp mono-detection and Mp co-detection were recorded and analyzed. RESULTS: Among the 110 CAP children with Mp positive, 42 (38.18%) of them were co-detected with at least one other pathogen. Co-detection was more common among children aged ≤3 years. No significant differences were found in most clinical symptoms, complications, underlying conditions and disease severity parameters among various etiological groups, with the following exceptions. First, prolonged duration of fever, lack of appetite and runny nose were more prevalent among CAP children with Mp-virus co-detection. Second, Mp-virus (excluding HRV) co-detected patients were more likely to present with prolonged duration of fever. Third, patients co-detected with Mp-bacteria were more likely to have abnormal blood gases. Additionally, CAP children with Mp-HRV co-detection were significantly more likely to report severe runny nose compared to those with Mp mono-detection. CONCLUSION: Mp co-detection with viral and/or bacterial pathogens is common in clinical practice. However, there are no apparent differences between Mp mono-detection and Mp co-detections in terms of clinical features and disease severity.
Subject(s)
Community-Acquired Infections/diagnosis , Mycoplasma pneumoniae/isolation & purification , Pneumonia, Mycoplasma/diagnosis , Bacteria/isolation & purification , Bronchoalveolar Lavage Fluid/microbiology , Child , Child, Hospitalized , Child, Preschool , Community-Acquired Infections/microbiology , DNA, Bacterial/metabolism , Female , Humans , Infant , Male , Mycoplasma pneumoniae/genetics , Pneumonia, Mycoplasma/microbiology , Prevalence , Prospective Studies , Viruses/isolation & purificationABSTRACT
BACKGROUND: Hepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices. METHODS: Here we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D). RESULTS: A total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818. CONCLUSIONS: We developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing.
Subject(s)
Hepatitis B virus/isolation & purification , Hepatitis B/diagnosis , Nucleic Acid Amplification Techniques , Recombinases/metabolism , Adult , DNA, Viral/isolation & purification , DNA, Viral/metabolism , Female , Hepatitis B/virology , Humans , Male , Middle Aged , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Thiols play a vital role in both the physiological process and organic synthesis field, including aliphatic thiols (e.g., Cys, Hcy, and GSH) and thiophenols. As a result of the similarities of thiols in terms of molecular structure and chemical properties, it is difficult for conventional fluorescent probes to distinguish them, which hinders the progress of biological and pathological research. Keeping this in mind, a dual-site and dual-excitation fluorescent probe (YY) was designed to distinguish among Cys, Hcy, and thiophenols by three different reaction paths. When excited at 470 nm, YY only exhibits a fluorescence OFF-ON response toward thiophenols. However, when excited at 453 nm, YY not only displays a fluorescence OFF-ON response toward Hcy and thiophenols (λem = 499 and 561 nm) but also presents a two-stage fluorescence response toward Cys, which possesses a fluorescence OFF-ON response in the first stage (λem = 501 nm) and then a fluorescence ON-OFF response in the second stage (λem = 556 nm). This specific fluorescence response indicates that YY has ability to overcome the above-mentioned challenge to achieve discriminative detection of Cys, Hcy, and thiophenols qualitatively, which promotes the study of thiols in the fields of physiology and pathology. Furthermore, cell-imaging studies show that YY can be applied to the imaging of exogenous Cys, Hcy, and thiophenols through two different emission channels.
Subject(s)
Cysteine/analysis , Fluorescent Dyes/chemistry , Phenols/analysis , Sulfhydryl Compounds/analysis , Water Pollutants, Chemical/analysis , Cysteine/analogs & derivatives , Fluorescent Dyes/chemical synthesis , HeLa Cells , Humans , Lakes/chemistry , Molecular Structure , Optical Imaging , Rivers/chemistryABSTRACT
BACKGROUND: Respiratory syncytial virus (RSV), human Rhinovirus (HRV) and human Metapneumo Virus (HMPV) are important viral pathogens causing acute respiratory tract infections in the hospitalized patients. Sensitive and accurate detection of RSV, HRV and HMPV is necessary for clinical diagnosis and treatment. RESULTS: A locked nucleic acid (LNA)-based multiplex closed one-tube nested real-time RT-PCR (mOTNRT-PCR) assay was developed for simultaneous detection of RSV, HRV and HMPV. The sensitivity, specificity, reproducibility and clinical performance of mOTNRT-PCR were evaluated and compared with individual real time PCR (RT-qPCR) assay using clinical samples. The analytical sensitivity of mOTNRT-PCR assay was 5 copies/reaction for RSV, HRV and HMPV, respectively, and no cross-reaction with other common respiratory viruses was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.51 to 3.67%. Of 398 nasopharyngeal aspirates samples tested, 109 (27.39%), 150 (37.69%) and 44 (11.06%) were positive for RSV, HRV and HMPV, respectively, whereas 95 (23.87%), 137 (34.42%) and 38 (9.55%) were positive for RSV, HRV and HMPV, respectively, by individual RT-qPCR assay. Thirty three samples that were positive by mOTNRT-PCR but negative by RT-qPCR were confirmed as true positives by sequencing using reported traditional two-step nested PCR assay. CONCLUSION: mOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings.
Subject(s)
Metapneumovirus/isolation & purification , Paramyxoviridae Infections/diagnosis , Picornaviridae Infections/diagnosis , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus, Human/isolation & purification , Rhinovirus/isolation & purification , Acute Disease , Child , Child, Preschool , Female , Humans , Infant , Male , Metapneumovirus/genetics , Multiplex Polymerase Chain Reaction , Nasopharynx/virology , Reproducibility of Results , Respiratory Syncytial Virus, Human/genetics , Rhinovirus/genetics , Sensitivity and SpecificityABSTRACT
BACKGROUND: Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. METHODS: In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. RESULTS: The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). CONCLUSION: The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.
Subject(s)
Adenoviruses, Human/genetics , Molecular Typing/methods , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , Adenovirus Infections, Human/virology , Adenoviruses, Human/classification , Adenoviruses, Human/isolation & purification , Child , Child, Preschool , Humans , Infant , Molecular Typing/instrumentation , Molecular Typing/standards , Multiplex Polymerase Chain Reaction/standards , Nasopharynx/virology , Observer Variation , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Respiratory Tract Infections/virology , Sensitivity and Specificity , SerogroupABSTRACT
BACKGROUND: Diarrhea is a major source of morbidity and mortality among young children in low-income and middle-income countries. Human adenoviruses (HAdV), particular HAdV species F (40, 41) has been recognized as important causal pathogens, however limited data exist on molecular epidemiology of other HAdV associated with acute gastroenteritis. METHODS: In the present preliminary study, we performed a case-control study involving 273 children who presented diarrheal disease and 361 healthy children matched control in Children's hospital of Hebei Province (China) to investigate the relationship between non-enteric HAdV and diarrhea. HAdV were detected and quantified using quantitative real-time PCR (qPCR) and serotyped by sequencing and phylogenetic analysis. Odds ratio (OR) was used to assess the risk factor of HAdV. RESULTS: HAdV were detected in 79 (28.94%) of 273 children with diarrhea including 7 different serotypes (HAdV 40, 41, 3, 2,1,5 and 57) with serotypes 40, 41 and 3 being the most dominant and in 26 (7.20%) of 361 healthy children containing 9 serotypes (HAdV 40, 41, 3, 2,1,5,57,6 and 31). A majority (91.14%) of HAdV positives occurred in diarrhea children and 65.38% in controls< 3 years of age. No significant difference in the viral load was found between case and control groups or between Ad41-positive patients and healthy controls. In addition to HAdV 40 and 41, HAdV 3 was also associated with diarrhea (OR = 17.301, adjusted OR = 9.205, p < 0.001). CONCLUSIONS: Our results demonstrate a high diversity of HAdV present among diarrhea and healthy children and implicate that non-enteric HAdV3 may lead to diarrhea.
Subject(s)
Adenovirus Infections, Human/epidemiology , Adenovirus Infections, Human/virology , Diarrhea/epidemiology , Diarrhea/virology , Adenovirus Infections, Human/complications , Adenoviruses, Human/genetics , Adenoviruses, Human/isolation & purification , Adolescent , Case-Control Studies , Child , Child, Preschool , China/epidemiology , Female , Gastroenteritis/epidemiology , Gastroenteritis/virology , Humans , Infant , Male , Molecular Epidemiology , Phylogeny , Real-Time Polymerase Chain Reaction , Serotyping , Viral LoadABSTRACT
OBJECTIVE: To investigate the association between single nucleotide polymorphisms( SNPs) of 5, 10-methylenetetrahydrofolate reductase( MTHFR) C677T, A1298C, methionine synthase( MS) A2756G, methionine synthase reductase( MTRR) A66G and the risk of congenital heart disease( CHD). METHODS: By restricting the corresponding conditions, a case-control study was performed. We collected 200 congenital heart disease children as case group and 200 normal children as control group admitted to the Department of cardiac surgery, Children 's Hospital of Hebei Province from January 2016 to April 2017. The genotype of MTHFR C677T, A1298C, MS A2756G, and MTRR A66G polymorphisms were detected by Sanger sequencing followed PCR. Assessing the relationships between 4 SNPs and the risk of whole CHD and different types of CHD. RESULTS: The mutant allete T of MTHFR C677T had contribute to the risk of developing CHD( OR = 2. 47, 95% CI 1. 86-3. 29, P < 0. 001). Compared with the wild CC genotype, heterozygosity CT had a higher risk of CHD( OR = 2. 32, 95% CI 1. 35-3. 98, P < 0. 05), the homozygous mutant genotype TT increased the risk of CHD by 5. 37( 95% CI 3. 01-9. 60, P < 0. 001). The mutant allele C of MTHFR A1298C was a protective factor for CHD( OR = 0. 53, 95% CI 0. 36-0. 77, P < 0. 05). Compared with the wild AA genotype, heterozygosity AC had a lower risk of CHD( OR = 0. 41, 95% CI0. 26-0. 64, P < 0. 001). After typing, the allele frequencies and genotypes frequencies of the above two SNPs were still statistically significant( P < 0. 05). The combined genotype analysis of the above two SNPs showed that: compared with the CC/AA genotype, individuals with CT/AA had a higher risk of CHD( OR = 4. 65, 95% CI 2. 16-10. 02), the TT/AA type increased to 7. 05( 95% CI 3. 37-14. 79). However, the polymorphisms of MS A2756G and MTRR A66G had no significant relationship with the risk of CHD( P > 0. 05). CONCLUSION: The mutant allele T of MTHFR C677T may be a risk factor for CHD and the mutant allele C of A1298C may be a protective factor for CHD. These two SNPs may have a joint effect on the occurrence of CHD.
Subject(s)
5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/genetics , Ferredoxin-NADP Reductase/genetics , Folic Acid/metabolism , Genetic Predisposition to Disease , Heart Defects, Congenital/genetics , Heart Defects, Congenital/metabolism , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , Polymorphism, Single Nucleotide , Case-Control Studies , Child , Genotype , Humans , Polymorphism, Genetic , Risk FactorsABSTRACT
To probe the kinetic performance of microsolvated α-nucleophile, the G2(+)M calculations were carried out for the gas-phase S(N)2 reactions of monohydrated and dihydrated α-oxy-nucleophiles XO(-)(H2O)(n = 1,2) (X = HO, CH3O, F, Cl, Br), and α-sulfur-nucleophile, HSS(-)(H2O)(n = 1,2), toward CH3Cl. We compared the reactivities of hydrated α-nucleophiles to those of hydrated normal nucleophiles. Our calculations show that the α-effect of monohydrated and dihydrated α-oxy-nucleophiles will become weaker than those of unhydrated ones if we apply a plot of activation barrier as a function of anion basicity. Whereas the enhanced reactivity of monohydrated and dihydrated ROO(-) (R = H, Me) could be observed if compared them with the specific normal nucleophiles, RO(-) (R = H, Me). This phenomena can not be seen in the comparisons of XO(-)(H2O)(n = 1,2) (X = F, Cl, Br) with ClC2H4O(-)(H2O)(n = 1,2), a normal nucleophile with similar gas basicity to XO(-)(H2O)(n = 1,2). These results have been carefully analyzed by natural bond orbital theory and activation strain model. Meanwhile, the relationships between activation barriers with reaction energies and the ionization energies of α-nucleophile are also discussed.
Subject(s)
Gases , Combinatorial Chemistry Techniques , Nitro Compounds/chemistry , Sulfur , Thermodynamics , WaterABSTRACT
The lysine(K)-specific methyltransferase 2A gene (KMT2A), previously known as mixed lineage leukemia (MLL), frequently rearranged in acute leukemia, belongs to one of the most promiscuous genes and has been found fused to more than 80 different partners. KMT2A::SEPTIN6 fusion is a relatively uncommon rearrangement observed in pediatric acute myeloid leukemia (AML) patients, some of which may harbor other mutations. We herein report a case of AML-M4-infant with KMT2A::SEPTIN6 fusion and DIS3 variant. The 8-month-old girl presented with leukocytosis, anemia and thrombocytopenia. A bone marrow smear disclosed that 64% of the total nucleated cells were blasts. Karyotype analysis showed 46,X,t(X;11)(q24;q23)[10]/46,XX[10]. Fluorescence in situ hybridization analysis suggested a possible break in the KMT2A gene. After whole transcriptome sequencing, Exon 9 of KMT2A was fused in-frame with Exon 2 of SEPTIN6. This is a typical type of chromosomal rearrangement leading to the KMT2A::SEPTIN6 fusion. Meanwhile, DIS3 variant [c.2065C>T, p.R689X, variant allele frequency (VAF): 39.8%] was identified. KMT2A::SEPTIN6 fusion has been associated with the pathogenesis of AML, whereas DIS3 variants are relatively rare genetic events in pediatric AML. Regrettably, the relatives disagreed with the combination chemotherapy, and the patient eventually died of progressive disease. In conclusion, our findings provide a foundation for a better understanding of the genotypic profile of KMT2A::SEPTIN6 associated AML, and the co-existence of KMT2A::SEPTIN6 and DIS3 variant might contribute to the disease progression and transformation of AML.
ABSTRACT
BACKGROUND: Human adenovirus (HAdV) is a common pathogen in children that can cause acute respiratory tract infection (ARTI), but the molecular epidemiological and clinical information relating to HAdV among hospitalized children with ARTI are few reported in China. OBJECTIVES: To evaluate the epidemiological, clinical, and molecular characteristics of HAdV infections among hospitalized children with ARTI in Hebei, Northern China from June 2017 to May 2018. STUDY DESIGN: A 12-month longitudinal, retrospective study on HAdV, typed by nested polymerase chain reaction targeting the hexon gene's hypervariable region (typing was merely performed by sequencing of the hexon neutralization epitope and thus genotypes could not be identified unequivocally), associated with ARTI was performed. The epidemiological and clinical data of different types of HAdV were analyzed using statistical product and service solutions (SPSS) 21.0 software. RESULTS: HAdV was detected in 330 (3.71%) of the 8906 specimens, with most (88.48%, 292/330) HAdV-positives cases detected among children < 3 years old. HAdV were detected throughout the year with a higher prevalence in spring. 11 types were identified, with HAdV-2 (33.33%, 110/330) as the predominant type, followed by HAdV-3 (21.21%, 70/330) and HAdV-7 (13.94%, 46/330). Of the 330 HAdV-positive specimens, 247 (74.85%) were co-detected with other respiratory pathogens, most commonly rhinovirus (HRV) (58.7%, 145/247). Additionally, patients with HAdV-7 positive had longer duration of fever than HAdV-2 or -3 positive patients. CONCLUSIONS: During the study period, HAdV-2, HAdV-3 and HAdV-7 were the predominant types identified from children with ARTI in Hebei Province. Pediatric patients with HAdV-7 positive may not present more severe clinical outcome except a longer duration of fever.
Subject(s)
Adenovirus Infections, Human/epidemiology , Adenoviruses, Human/genetics , Hospitalization/statistics & numerical data , Respiratory Tract Infections/virology , Acute Disease/epidemiology , Adenovirus Infections, Human/virology , Adenoviruses, Human/classification , Adenoviruses, Human/pathogenicity , Adolescent , Child , Child, Preschool , China/epidemiology , Coinfection/epidemiology , Coinfection/virology , DNA, Viral/genetics , Female , Genotype , Humans , Infant , Longitudinal Studies , Male , Phylogeny , Prevalence , Respiratory Tract Infections/epidemiology , Retrospective Studies , Sequence Analysis, DNAABSTRACT
Cys is one of the important biothiols and its abnormal concentration may pose a threat to human health. Therefore, the monitoring of Cys in organisms is of great significance. GSH and Hcy, as the other two biothiols, have similar chemical structures and active sites to Cys. Consequently, developing fluorescent probes to independently detect Cys has become a challenging problem. Keeping this in mind, α-ß unsaturated ketone as a recognition group was integrated into the coumarin group skeleton to synthesize a fluorescent probe SC. After the nucleophilic addition reaction of Cys with SC, the conjugated system of SC was blocked and the fluorescent enhanced obviously. SC was able to detect Cys specifically under the same excitation with a low detection limit (11.1â¯nM). SC showed a rapid respond to Cys (120â¯s) and good fluorescent stability over a wide pH range. In addition, it achieved extracorporeal circulation in the presence of H2O2 or NEM. In the end, SC could be applied to detecting endogenous and exogenous Cys under biological condition due to its slight cytotoxicity and good biocompatibility. This provided a powerful tool for studying the physiological function of Cys exclusively.
Subject(s)
Coumarins/chemistry , Cysteine/analysis , Fluorescent Dyes/chemistry , Coumarins/chemical synthesis , Coumarins/toxicity , Cysteine/chemistry , Ethylmaleimide/chemistry , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/toxicity , HeLa Cells , Humans , Hydrogen Peroxide/chemistry , Hydrogen-Ion Concentration , Limit of Detection , Microscopy, Confocal/methods , Microscopy, Fluorescence/methodsABSTRACT
Hydrogen sulfide (H2S) plays a vital role in numerous biological processes in living organisms. To better understand its functions, a fluorescent probe to fast and sensitively detect H2S is imminently needed. Keep this in mind, we reasonably designed probe DHC for detecting H2S based on α, ß-unsaturated ethanoylcoumarin fluorophore. The limit of detection (LOD) is found to be as low as 5â¯×â¯10-8 M, which is superior to most reported fluorescent probes to detect H2S. Furthermore, the wide pH range of 4-11 makes it capable of application in biological systems. Most importantly, MTT assays and cell imaging experiments indicate that probe DHC has hypotoxicity and outstanding membrane permeability, which makes DHC successful imaging of H2S in Baby Hamster Syrian Kidney (BHK) cells.
Subject(s)
Colorimetry , Coumarins/chemistry , Fluorescent Dyes/chemistry , Hydrogen Sulfide/analysis , Animals , Cell Line , Cell Survival/drug effects , Coumarins/chemical synthesis , Coumarins/pharmacology , Cricetinae , Dose-Response Relationship, Drug , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/pharmacology , Molecular Structure , Optical Imaging , Structure-Activity RelationshipABSTRACT
Respiratory syncytial virus (RSV) causes serious respiratory tract infection worldwide. The relatively low RSV load makes it difficult to detect in frail, elderly, and severely immune-compromised patients. In the present study, we developed a locked nucleic acid--based 1-tube nested real-time RT-PCR (OTNRT-PCR) assay with the advantages of extremely high sensitivity, facile operability, and less likelihood of cross-contamination. The sensitivity, specificity, and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional 2-step nested RT-PCR assay. The limit of detection of the OTNRT-PCR assay was 1.02â¯×â¯10-1 TCID50/mL, equivalent to the traditional 2-step nested RT-PCR assay and 25-fold lower than the qRT-PCR assay. Of 616 nasopharyngeal aspirates tested, 143 RSV-negative samples by qRT-PCR were confirmed as positive by sequencing the OTNRT-PCR products. We therefore conclude that OTNRT-PCR is more sensitive than qRT-PCR for detection of RSV in clinical samples.
Subject(s)
Oligonucleotides/genetics , Real-Time Polymerase Chain Reaction/methods , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus, Human/genetics , DNA Primers/genetics , Humans , Limit of Detection , Nucleic Acid Denaturation , RNA, Viral/analysis , RNA, Viral/genetics , Respiratory Syncytial Virus, Human/isolation & purificationABSTRACT
A novel 2D lanthanide coordination polymer {Eu-CP (1)} based on a 3-bis(3-carboxyphenyl)imidazolium (L) ligand was successfully assembled through a solvothermal method. Luminescence tests indicate that 1 has excellent selectivity and sensitivity to detect Fe3+/Fe2+, Cr2O72-, and a series of nitroaromatic explosives. Interestingly, 1 can effectively distinguish CrVI anions (CrO42-/Cr2O72-), which is difficult for most of the reported CPs. Furthermore, in the detection process, 1 can be simply and quickly regenerated and reused at least five times. As far as we know, 1 is the first multifunctional Eu-CP used as a luminescent probe to detect Fe3+/Fe2+, Cr2O72-, and a series of nitroaromatic explosives (NAEs) based on the imidazolium derivative.
ABSTRACT
By choosing 2-pyridin-4-yl-4,5-imidazoledicarboxylic acid (H3PIDC) as the first ligand and sodium oxalate (OX) as the ancillary ligand, a series of mixed-lanthanide metal-organic frameworks (M'LnMOFs) [Tb1-xEux(HPIDC)(ox)1/2H2O]·3H2O (x = 0 1, 0.01 2a, 0.03 2b, 0.05 2c, 0.08 2d, 0.1 2e, 0.3 2f, 0.5 2g, 1 3) have been successfully synthesized via hydrothermal reactions. 2a-2f can serve as ratiometric luminescent sensors for detecting temperature. In this co-doped system, 2d shows an excellent linear response relationship with temperature from 303 to 473 K and exhibits a maximum relative sensitivity (Sr) of 0.60% K-1 at 473 K. Furthermore, powder X-ray diffraction (PXRD) experiments indicate that 2d has excellent chemical stability under simulated physiological conditions and alkali-acid solutions with pH ranging from 4 to 11, which makes it suitable to be applied in the physiological environment.
ABSTRACT
Single nucleotide polymorphisms (SNPs) play an important role in susceptibility to complex diseases, treatment efficacy and adverse drug responses. Conventional methods to detect SNPs are usually based on PCR or DNA sequencing, which are typically time-consuming and require sophisticated equipment. In this proof-of-concept study, a probe-directed recombinase amplification (PDRA) assay was developed to detect the A1298C polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR). The PDRA assay included two real-time reactions to detect the A and C nucleotides of A1298C polymorphism. Each reaction contained only one primer and one probe and was finished at 39°C within 35 min. The results of genotyping of 150 clinical samples using PDRA were completely consistent with those by direct sequencing. Additionally, when the 1000 Genomes Project HCB frequencies were used as the control group, MTHFR A1298C was found to be associated with congenital heart disease. In conclusion, the proposed novel PDRA assay is a valuable tool for the detection of SNPs and demonstrates significant potential to be widely applicable in both research and clinical settings.
Subject(s)
Heart Defects, Congenital/genetics , Methylenetetrahydrofolate Reductase (NADPH2)/chemistry , Nucleic Acid Amplification Techniques , Polymorphism, Single Nucleotide , Genetic Predisposition to Disease , Genotype , Genotyping Techniques , Humans , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , Proof of Concept StudyABSTRACT
The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10-8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2-10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples.