Targeting tau only extracellularly is likely to be less efficacious than targeting it both intra- and extracellularly.

Aggregation of the tau protein is thought to be responsible for the neurodegeneration and subsequent functional impairments in diseases that are collectively named tauopathies. Alzheimer's disease is the most common tauopathy, but the group consists of over 20 different diseases, many of which have tau pathology as their primary feature. The development of tau therapies has mainly focused on preventing the formation of and/or clearing these aggregates. Of these, immunotherapies that aim to either elicit endogenous tau antibodies or deliver exogenous ones are the most common approach in clinical trials. While their mechanism of action can involve several pathways, both extra- and intracellular, pharmaceutical companies have primarily focused on antibody-mediated clearance of extracellular tau. As we have pointed out over the years, this is rather surprising because it is well known that most of pathological tau protein is found intracellularly. It has been repeatedly shown by several groups over the past decades that antibodies can enter neurons and that their cellular uptake can be enhanced by various means, particularly by altering their charge. Here, we will briefly describe the potential extra- and intracellular mechanisms involved in antibody-mediated clearance of tau pathology, discuss these in the context of recent failures of some of the tau antibody trials, and finally provide a brief overview of how the intracellular efficacy of tau antibodies can potentially be further improved by certain modifications that aim to enhance tau clearance via specific intracellular degradation pathways.

[Genetic characterization and drug resistance analysis of Salmonella Kentucky ST314 in Shenzhen in 2010-2021].

OBJECTIVE: To understand the prevalence, genetic characteristics and drug resistance features of Salmonella Kentucky ST314 in Shenzhen. METHODS: Whole genome sequencing of 14 strains of Salmonella Kentucky ST314 collected from 2010-2021 by the Foodborne Disease Surveillance Network of Shenzhen Center for Disease Control and Prevention for phylogenetic evolutionary analysis, drug resistance gene and plasmid detection; drug susceptibility experiments were performed by micro-broth dilution method. RESULTS: A total of 57 strains of Salmonella Kentucky were collected from the foodborne disease surveillance network, 14 of which were ST314. The Shenzhen isolates were clustered with isolates from Southeast Asian countries such as Vietnam and Thailand on clade 314.2, and the single nucleotide polymorphism distance between local strains in Shenzhen was large, indicating dissemination. In this study, a total of 17 drug resistance genes/mutations in 9 categories were detected in the genome of Salmonella Kentucky ST314, carrying 3 extended spectrum beta-lactamases(ESBLs), including bla_(CTX-M-24)(14.3%, 2/14), bla_(CTX-M-55)(7.1%, 1/14), and bla_(CTX-M-130)(14.3%, 2/14), all located on plasmids. Regarding quinolone resistance factors, two plasmid-mediated quinolone resistance(PMQR) genes were identified in the genome: qnrB6(71.4%, 10/14) and aac(6')Ib-cr(78.6%, 11/14), a quinolone resistance quinolone resistance-determining regions(QRDR) mutation T57 S(100%, 14/14). The multi-drug resistance rate of Salmonella Kentucky ST314 in Shenzhen was 92.86%(13/14)with the highest rate of resistance to tetracycline and cotrimoxazole(100%, 14/14), followed by chloramphenicol(92.86%, 13/14), cefotaxime and ampicillin(78.57%, 11/14), ciprofloxacin and nalidixic acid(71.43%, 10/14), and ampicillin-sulbactam had the lowest resistance rate(21.43%, 3/14). CONCLUSION: ST314 is the second most prevalent ST type among Salmonella Kentucky in Shenzhen, mainly isolated from food, especially poultry; phylogenetic analysis suggests that ST314 is a disseminated infection and the genome shows a highly genetically conserved phenotype. Drug resistance of Salmonella Kentucky ST314 is very serious, especially QRDR mutation, PMQR gene co-mediated quinolone resistance and plasmid-mediated cephalosporin resistance are prominent and deserve extensive attention.

The utility of diffusion-weighted imaging and ADC values in the characterization of mumps orchitis and seminoma.

BACKGROUND: Diffusion-weighted imaging (DWI) can quantitatively reflect the diffusion characteristics of tissues, providing a theoretical basis for qualitative diagnosis and quantitative analysis of a disease. PURPOSE: To characterize testicular lesions that present as a hypointense signal on magnetic resonance imaging (MRI) T2-weighted images using DWI. MATERIAL AND METHODS: Study participants were divided into three groups. Group A were healthy controls (n = 35), group B included patients with mumps orchitis (n = 20), and group C included patients with seminoma (n = 15). DWI sequences used b-values of 0, 1000, and 2000 s/mm2. Apparent diffusion coefficient (ADC) values between 1000 and 2000 s/mm2 were calculated by MRI postprocessing software. The Kruskal-Wallis test and receiver operating characteristic analysis were performed to evaluate how well ADC values distinguished between mumps orchitis and seminoma. RESULTS: Normal testicular tissue showed a hyperintense signal on DWI and hypointensity on the ADC map: mean ADC value was 0.77 (0.69-0.85) ± 0.08 ×10-3 mm2/s. Mumps orchitis and seminoma showed slight hyperintensity on DWI: mean ADC values were 0.85 (0.71-0.99) ± 0.15 ×10-3 mm2/s and 0.43 (0.39-0.47) ± 0.04 × 10-3 mm2/s, respectively. There were statistically significant differences in mean ADC values between normal testicular tissue and seminoma and between mumps orchitis and seminoma. The cutoff ADC value for differentiating seminoma from mumps orchitis was 0.54 × 10-3 mm2/s. The sensitivity, specificity, and Youden Index for diagnosing seminoma were 99%, 31%, and 30%, respectively. CONCLUSION: High b-value DWI has potential utility for differentiating mumps orchitis from seminoma in the clinical setting.

Investigation and Identification of Food Poisoning Caused by Clostridium botulinum Type B1 in Shenzhen, China.

Clostridium botulinum produces botulinum neurotoxins (BoNTs), which cause people who ingest them to become seriously ill and sometimes die. In recent years, sporadic food poisoning cases associated with C. botulinum have occurred across the world. In 2016, two men were admitted to our hospital in Shenzhen, China, with foodborne botulism. In this study, we report on these two typical C. botulinum-related food poisoning incidents and the steps taken to identify and characterize the causative pathogen. We characterized the bacterial pathogen isolated from the first patient using cooked meat medium and egg yolk agar bacterial cultures under anaerobic conditions, and morphologically identified the isolate using Gram staining. The in vivo bioassay results in mice showed that the minimum lethal dose of the BoNTs produced by our isolate was 0.001-0.0001 mg/mL (LD50 of the culture was estimated to be 1.5812 mg/kg). Whole genome sequencing (WGS) results showed that the isolate was identified as C. botulinum B1 Okra. The causative strain was successfully isolated from the intestinal lavage fluid collected from the initial patient.

Concise solid-phase synthesis enables derivatisation of YEATS domain cyclopeptide inhibitors for improved cellular uptake.

YEATS domains, which are newly identified epigenetic readers of histone lysine acetylation and crotonylation, have emerged as promising anti-cancer drug targets. We recently developed AF9 YEATS domain-selective cyclopeptide inhibitors. However, the cumbersome and time-consuming synthesis of the cyclopeptides limited further structural derivatisation and applications. Here, we reported a concise method for the solid-phase synthesis of the cyclopeptides, which substantially reduced the amount of time required for the preparation of the cyclopeptides and led to a higher overall yield. Moreover, this new synthetic route also allowed further derivatisation of the cyclopeptides with various functional modules, including fluorescent dye and cell-penetrating peptide. We demonstrated that the conjugation of the cyclopeptide with cell-penetrating peptide TAT led to a significantly increased cellular uptake.

Comparative evaluation of six nucleic acid amplification kits for SARS-CoV-2 RNA detection.

BACKGROUND: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B-F) to evaluate their sensitivity, specificity, predictive values and accuracy. METHODS: Fifty-two clinical samples were used including throat swabs (n = 30), nasal swabs (n = 7), nasopharyngeal swabs (n = 7) and sputum specimens (n = 8), comprising confirmed (n = 26) and negative cases (n = 26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits' evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. RESULTS: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa ≥ 0.75); Kits D and E were less congruent (0.4 ≤ Kappa < 0.75). Differences between all kits were statistically significant (P < 0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. CONCLUSIONS: This is the first comparative study to evaluate CPA and RT-qPCR kits' specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic.

Targeting the Amyloid-ß Fibril Surface with a Constrained Helical Peptide Inhibitor.

Amyloid-ß (Aß) oligomers are well-known toxic molecular species associated with Alzheimer's disease. Recent discoveries of the ability of amyloid fibril surfaces to convert soluble proteins into toxic oligomers suggested that these surfaces could serve as therapeutic targets for intervention. We have shown previously that a short helical peptide could be a key structural motif that can specifically recognize the K16-E22 region of the Aß40 fibril surface with an affinity at the level of several micromolar. Here, we demonstrate that in-tether chiral center-induced helical stabilized peptides could also recognize the fibril surfaces, effectively inhibiting the surface-mediated oligomerization of Aß40. Moreover, through extensive computational sampling, we observed two distinct ways in which the peptide inhibitors recognize the fibril surface. Apart from a binding mode that, in accord with the original design, involves hydrophobic side chains at the binding interface, we observed much more frequently another binding mode in which the hydrophobic staple interacts directly with the fibril surface. The affinity of the peptides for the fibril surface could be adjusted by tuning the hydrophobicity of the staple. The best candidate investigated here exhibits a submicromolar affinity (∼0.75 µM). Collectively, this work opens an avenue for the rational design of candidate drugs with stapled peptides for amyloid-related disease.

Selective Targeting of AF9 YEATS Domain by Cyclopeptide Inhibitors with Preorganized Conformation.

YEATS domains are newly identified epigenetic "readers" of histone lysine acetylation (Kac) and crotonylation (Kcr). The malfunction of YEATS-Kac/Kcr interactions has been found to be involved in the pathogenesis of human diseases, such as cancer. These discoveries suggest that the YEATS domains are promising novel drug targets. We and others recently reported the development of YEATS domain inhibitors. Although these inhibitors have a general preference toward the AF9 and ENL YEATS domains, selective inhibitors targeting either YEATS domain are challenging to develop as these two proteins share a high structural similarity. In this study, we identified a proximal site outside the acyllysine-binding pocket that can differentiate AF9 YEATS from ENL YEATS. Combinatorial targeting of both the acyllysine pocket and this additional site by conformationally preorganized cyclopeptides enabled the selective inhibition of the AF9 YEATS domain. The most selective inhibitor, JYX-3, showed a 38-fold higher binding affinity toward AF9 YEATS over ENL YEATS. Further investigations indicated that JYX-3 could engage with AF9 in living cells, disrupt the YEATS-dependent chromatin recruitment of AF9, and suppress the transcription of AF9 target genes.

Structure-guided development of YEATS domain inhibitors by targeting π-π-π stacking.

Chemical probes of epigenetic 'readers' of histone post-translational modifications (PTMs) have become powerful tools for mechanistic and functional studies of their target proteins in normal physiology and disease pathogenesis. Here we report the development of the first class of chemical probes of YEATS domains, newly identified 'readers' of histone lysine acetylation (Kac) and crotonylation (Kcr). Guided by the structural analysis of a YEATS-Kcr complex, we developed a series of peptide-based inhibitors of YEATS domains by targeting a unique π-π-π stacking interaction at the proteins' Kcr recognition site. Further structure optimization resulted in the selective inhibitors preferentially binding to individual YEATS-containing proteins including AF9 and ENL with submicromolar affinities. We demonstrate that one of the ENL YEATS-selective inhibitors, XL-13m, engages with endogenous ENL, perturbs the recruitment of ENL onto chromatin, and synergizes the BET and DOT1L inhibition-induced downregulation of oncogenes in MLL-rearranged acute leukemia.

[Rapid identification of 15 common S. enterica Salmonella serovars by multiplex real-time PCR].

OBJECTIVE: Multiplex real-time PCR for the identification of 15 Salmonella serovars was developed. METHODS: Through the Salmonella genome comparison, 12 membrane proteins STM4497 gene can be used to identify 15 Salmonella serovars, and these 12 genes were respectively listed as A-L genes. Then primers were designed according to A-L gene conserved sequences, and then multiplex real-time PCR was established assessed with the evaluation of the limit detection, sensitivity, specificity, and repeatability. The 206 Salmonella strains were identified using multiplex real-time PCR with the comparison of the serum slide agglutination assay. RESULTS: The limit detection of multiplex PCR ranged from 1. 1×10~(-3)-1. 2×10~(-3) ng/µL. The target genes were 100% specificity, and the relative standard deviation was lower than 2. 97%. Compared with the serum slide agglutination assay, Kappa ranged 0. 92-1. 00. CONCLUSION: The multiplex real-time PCR can be used to identify 15 Salmonella serovars, which is rapid, accurate and specific.

Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells.

Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.

Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars.

BACKGROUND: While Salmonella serotyping is of paramount importance for the disease intervention of salmonellosis, a fast and easy-to-operate molecular serotyping solution remains elusive. We have developed a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the identification of 30 common Salmonella serovars. METHODS: Serovar-specific primers and probes were designed based on a comparison of gene targets (wzx and wzy encoding for somatic antigen biosynthesis; fliC and fljB for flagellar antigens) from 5868 Salmonella genomes. The ssaR gene, a type III secretion system component, was included for the confirmation of Salmonella. RESULTS: All gene targets were detected and gave expected Tm values during assay evaluation. Cross reactions were not demonstrated between the 30 serovars (n = 211), or with an additional 120 serovars (n = 120) and other Enterobacteriaceae (n = 3). The limit of identification for all targets ranged from using 1.2 ng/µL to 1.56 ng/µL of DNA. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 0.5 °C and less than 1% respectively, indicating high reproducibility. From consecutive outpatient stool samples (n = 3590) collected over a 10-month period at 11 sentinel hospitals in Shenzhen, China, we conducted a multicenter study using the traditional Salmonella identification workflow and the MLMA assay workflow in parallel. From Salmonella isolates (n = 496, 13.8%) derived by both workflows, total agreement (kappa = 1.0) between the MLMA assay and conventional serotyping was demonstrated. CONCLUSIONS: With an assay time of 2.5 h, this simple assay has shown promising potential to provide rapid and high-throughput identification of Salmonella serovars for clinical and public health laboratories to facilitate timely surveillance of salmonellosis.

Dual In-Tether Chiral Centers Modulate Peptide Helicity.

The facile chemical modification on the peptide cross-linking moiety is an important strategy for improving the physicochemical properties of a peptide. Herein, peptides were constrained into helical conformations via the synergistic effects of dual in-tether chiral centers. A pentapeptide minimalistic model was used to determine the correlation between the absolute configurations of the dual in-tether chiral centers and the secondary structures of the peptides. This strategy provides an on-tether modification site that does not interrupt the secondary structure of the peptide.

Switching substitution groups on the in-tether chiral centre influences backbone peptides' permeability and target binding affinity.

Different substitution groups on the in-tether chiral centre of chirality-induced helical peptides (CIH peptides) showed distinguishable effects on the peptides' cellular uptakes and binding affinities with the estrogen receptor α(ER-α). This study proves that in-tether chiral centres are a valuable modification site for constructing peptide ligands with preferable biophysical properties.

Phenotypic and Genotypic Characterization of Clinical Enterotoxigenic Escherichia coli Isolates from Shenzhen, China.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of infectious diarrhea in developing countries. This study aimed to characterize the prevalence and phenotypic and genotypic features of ETEC isolates from Shenzhen, China. METHODS: ETEC isolates were obtained from acute diarrheal patients and evaluated for enterotoxin, classical colonization factors (CFs), serotypes, antimicrobial susceptibility, and multilocus sequencing typing (MLST). RESULTS: A total of 168 (1.3%) ETEC strains were isolated from 13,324 diarrheal outpatients during 2009 and 2014. A vast majority of ETEC-infected patients (82.1%) belonged to the age ranging 20-59 years and only six patients were children aged <5 years. Heat-stable toxin (ST) was most frequently detected (81.5%), followed by heat-labile toxin (LT) (13.1%). One or multiple colonization factors (CFs) were identified in 91 ETEC strains (54.2%). The most frequently detected CF was CS6 (with or without other CFs) (84/91), followed by CS21 (14/91). The most common serotype was O159:H34 (n = 36), followed by O148:H28 (n = 25) and O27:H7 (n = 17). High resistant rate was observed to nalidixic acid (77.4%), cephalothin (41.7%), ampicillin (34.5%), and tetracycline (21.4%). Antimicrobial resistance profiles differed among different serogroups. Sequence type (ST) 10 complex, integrated with connected ST218, ST48, ST4, and ST1312 subgroups, covered 73 (43.5%) isolates. CONCLUSIONS: ETEC isolates in Shenzhen of China appeared highly diverse, yet some isolates belonged to well-defined clonal groups sharing a unique set of virulence factors, serotypes, and MLST sequence types. Facing the challenge of ETEC antigenic diversity and geographic variation, novel molecules and/or classical antigens designed by novel strategies might contribute to ETEC vaccine development.

An in-tether sulfilimine chiral center induces ß-turn conformation in short peptides.

A sulfilimine chiral center in the tether at i, i + 3 positions of short peptides was systematically studied to elucidate the chirality-driven conformational changes. A rare and unexpected type III ß-turn structure was induced in short peptides by an in-tether chiral center, supported by circular dichroism spectroscopy, NMR and X-ray crystallography.

The evaluation and application of multilocus variable number tandem repeat analysis (MLVA) for the molecular epidemiological study of Salmonella enterica subsp. enterica serovar Enteritidis infection.

BACKGROUND: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the most prevalent Salmonella serotypes that cause gastroenteritis worldwide and the most prevalent serotype causing Salmonella infections in China. A rapid molecular typing method with high throughput and good epidemiological discrimination is urgently needed for detecting the outbreaks and finding the source for effective control of S. Enteritidis infections. METHODS: In this study, 194 strains which included 47 from six outbreaks that were well-characterized epidemiologically were analyzed with pulse field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA). Seven VNTR loci published by the US Center for Disease Control and Prevention (CDC) were used to evaluate and develop MLVA scheme for S. Enteritidis molecular subtyping by comparing with PFGE, and then MLVA was applied to the suspected outbreaks detection. All S. Enteritidis isolates were analyzed with MLVA to establish a MLVA database in Shenzhen, Guangdong province, China to facilitate the detection of S. Enteritidis infection clusters. RESULTS: There were 33 MLVA types and 29 PFGE patterns among 147 sporadic isolates. These two measures had Simpson indices of 0.7701 and 0.8043, respectively, which did not differ significantly. Epidemiological concordance was evaluated by typing 47 isolates from six epidemiologically well-characterized outbreaks and it did not differ for PFGE and MLVA. We applied the well established MLVA method to detect two S. Enteritidis foodborne outbreaks and find their sources successfully in 2014. A MLVA database of 491 S. Enteritidis strains isolated from 2004 to 2014 was established for the surveillance of clusters in the future. CONCLUSIONS: MLVA typing of S. Enteritidis would be an effective tool for early warning and epidemiological surveillance of S. Enteritidis infections.

Feasibility of Using Multiple-Locus Variable-Number Tandem-Repeat Analysis for Epidemiology Study of Vibrio parahaemolyticus Infections.

Vibrio parahaemolyticus causes foodborne gastroenteritis, which is often associated with the consumption of raw or undercooked shellfish. Molecular typing can provide critical information for detecting outbreaks and for source attribution. In this study, we describe the development and evaluation of an optimized multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) for the characterization of V. parahaemolyticus isolates. The discriminatory power of MLVA was compared to that of pulsed-field gel electrophoresis (PFGE) by typing 73 sporadic isolates. Epidemiologic concordance was evaluated by typing 23 isolates from five epidemiologically well-characterized outbreaks. The optimized MLVA was applied in early warning, epidemiological surveillance, and source tracking for V. parahaemolyticus infections. There was no significant difference in the discriminatory power of PFGE and MLVA with six or eight VNTR loci for the sporadic isolates. All isolates within an outbreak were indistinguishable by MLVA with six loci, except for one outbreak. Typically, the epidemiological survey could be initiated according to PFGE clusters. We applied MLVA with six loci on 22 isolates in two PFGE clusters. Isolates in one PFGE cluster were distinguished by MLVA. Although a follow-up investigation showed that both clusters had no epidemiological concordance, MLVA decreased the frequency of initiation of epidemiological surveys, thereby reducing labor costs. The ability of MLVA to trace the source of infection was evaluated by isolates from two outbreaks and shrimp samples. The isolates from one of outbreaks and a shrimp had the same MLVA type, suggesting that an epidemiological survey was initiated. Data from the epidemiological investigation subsequently indicated that contaminated shrimp from a nearby city (Dongguan) might be the source of the outbreak. In conclusion, these results indicate that the optimized MLVA may be a promising tool for early warning and epidemiological surveillance of V. parahaemolyticus infections.

Simultaneous detection of five enteric viruses associated with gastroenteritis by use of a PCR assay: a single real-time multiplex reaction and its clinical application.

We developed a highly sensitive reverse transcription and multiplex real-time PCR (rtPCR) assay that can identify five viruses, including six genogroups, in a single reaction: norovirus genogroups I and II; sapovirus genogroups I, II, IV, and V; human rotavirus A; adenovirus serotypes 40 and 41; and human astrovirus. In comparison to monoplex rtPCR assays, the sensitivities and specificities of the multiplex rtPCR ranged from 75% to 100% and from 99% to 100%, respectively, evaluated on 812 clinical stool specimens.

Rapid genetic typing of diarrheagenic Escherichia coli using a two-tube modified molecular beacon based multiplex real-time PCR assay and its clinical application.

BACKGROUND: Diarrheagenic Escherichia coli (DEC), including Enterotoxigenic E.coli (ETEC), Enteroaggregative E.coli (EAEC), Enteropathogenic E.coli (EPEC), Enterohemolysin E.coli (EHEC) and Enteroinvasive E.coli (EIEC) causes diarrhea or hemolytic uremic syndromes among infants and travelers around the world. A rapid, reliable and repeatable method is urgent for identifying DEC so as to provide the reference for responding to diarrheal disease outbreak and the treatment of the diarrheal patients associated with DEC. METHODS: In this study, specific primers and modified molecular beacon probes of nine specific virulence genes, whose 5'end were added with homo tail sequence, were designed; and a two-tube modified molecular beacon based multiplex real-time PCR (rtPCR) assay for the identification of five Escherichia coli pathotypes, including ETEC, EAEC, EPEC, EHEC and EIEC was developed and optimized. Totally 102 bacterial strains, including 52 reference bacterial strains and 50 clinical strains were detected to confirm whether the target genes selected were specific. Then detection limits of the assay were tested. Lastly, the assay was applied to the detection of 11860 clinical samples to evaluate the specificity and sensitivity of the developed assay compared with the conventional PCR. RESULTS: The target genes were 100% specific as assessed on 102 bacterial strains since no cross-reactions were observed. The detection limits ranged from 88 CFU/mL (EHEC) to 880 CFU/mL (EPEC). Compared with the conventional PCR, the specificity and sensitivity of the multiplex rtPCR was 100% and over 99%, respectively. The coefficient of variation (CV) for each target gene ranged from 0.45% to 1.53%. 171 positive clinical samples were mostly identified as ETEC (n = 111, 64.9%) and EPEC (n = 38, 22.2%), which were the dominating pathotypes of DEC strains. CONCLUSION: The developed multiplex rtPCR assay for the identification of DEC was high sensitive and specific and could be applied to the rapid identification of DEC in clinical and public health laboratories.