Infectious Disease Diagnosis and Pathogen Identification Platform Based on Multiplex Recombinase Polymerase Amplification-Assisted CRISPR-Cas12a System.

Controlling and mitigating infectious diseases caused by multiple pathogens or pathogens with several subtypes require multiplex nucleic acid detection platforms that can detect several target genes rapidly, specifically, sensitively, and simultaneously. Here, we develop a detection platform, termed Multiplex Assay of RPA and Collateral Effect of Cas12a-based System (MARPLES), based on multiplex nucleic acid amplification and Cas12a ssDNase activation to diagnose these diseases and identify their pathogens. We use the clinical specimens of hand, foot, and mouth disease (HFMD) and influenza A to evaluate the feasibility of MARPLES in diagnosing the disease and identifying the pathogen, respectively, and find that MARPLES can accurately diagnose the HFMD associated with enterovirus 71, coxsackievirus A16 (CVA16), CVA6, or CVA10 and identify the exact types of H1N1 and H3N2 in an hour, showing high sensitivity and specificity and 100% predictive agreement with qRT-PCR. Collectively, our findings demonstrate that MARPLES is a promising multiplex nucleic acid detection platform for disease diagnosis and pathogen identification.

Noninvasive Evaluation of Fetal Zygosity in Twin Pregnancies Involving a Binary Analysis of Single-Nucleotide Polymorphisms.

Twin pregnancy constitutes significant risks for maternal and fetal health, which is usually detected by ultrasound examination at early gestation. However, the imaging-based approach may not accurately identify all twins confounded by practical or clinical variables. The analysis of fetal cell-free DNA in noninvasive prenatal screening assays can completement the ultrasound method for twin detection, which differentiates fraternal or identical twins based on their distinct genotypes. Here, a new noninvasive prenatal screening employing high-coverage next-generation sequencing for targeted nucleotide polymorphisms was developed for detection of zygosity and determination of fetal fraction in twin pregnancies. This method utilizes a binary analysis of both the number and allelic fraction of fetus-specific single-nucleotide polymorphisms to infer the zygosity. In 323 samples collected from 215 singleton, 90 dizygotic, and 18 monozygotic twin pregnancies, all 90 dizygotic twins were correctly detected, with a 100% sensitivity and a 100% specificity. In addition, this method can detect complex pregnancies, such as egg donors, contamination, and twins with complete hydatidiform mole. The fetus-specific fetal fraction change was monitored in nine dizygotic twin pregnancies, which demonstrated highly variable dynamics of fetal cell-free DNA turnover up to 7 weeks after twin reduction. Overall, this study provides a new noninvasive prenatal screening strategy for the accurate identification of twin zygosity and quantification of fetal fraction, which has important clinical implications for the management of twin pregnancies.

Mitofusin 1-Mediated Redistribution of Mitochondrial Antiviral Signaling Protein Promotes Type 1 Interferon Response in Human Cytomegalovirus Infection.

One of the most potent anti-human cytomegalovirus (HCMV) immune mechanisms possessed by host cells is type I interferon (IFN1), which induces the expression of IFN-stimulated genes (ISGs). During this process, mitochondria play an important role in the IFN1 response, and mitofusin 1 (MFN1) is a key regulator of mitochondrial fusion located on the outer mitochondrial membrane. However, the underlying mechanism of MFN1's promotion of IFN1 during HCMV infection still remains unknown. In this study, HCMV infection promoted IFN1 production and enhanced ISG expression. Meanwhile, it promoted the increase of mitochondrial fusion in THP-1 cells and peripheral blood mononuclear cells (PBMCs), especially the expression of MFN1. Phosphorylation of tank binding kinase 1 (p-TBK1), interferon regulatory factor 3 (p-IRF3), and ISGs was significantly decreased in MFN1 or mitochondrial antiviral signaling protein (MAVS)-knockdown THP-1 cells, and MFN1 was constitutively associated with MAVS, positively regulated mitochondrial fusion, and IFN1 production. Knockdown of MFN1 inhibited the MAVS redistribution without affecting the MAVS expression, whereas the HCMV-induced IFN1 production decreased. Conversely, leflunomide could induce the expression of MFN1, thereby producing IFN1 and stimulating the expression of ISG in leflunomide-treated THP-1 cells. These observations reveal that HCMV infection leads to MFN1-mediated redistribution of MAVS and then induces an antiviral response of IFN1 and that the MFN-agonist leflunomide promotes IFN1 responses and may serve as a potential anti-HCMV therapy. IMPORTANCE Human cytomegalovirus (HCMV) infection is ubiquitous and is often asymptomatic in healthy individuals, but it can cause great damage to newborns, AIDS patients, and other immune deficiency patients. In this study, we found that HCMV infection caused mitochondrial fusion, and expression of mitofusin 1 (MFN1), which is a protein associated with mitochondrial antiviral signaling protein (MAVS), positively regulates mitochondrial fusion and HCMV-induced IFN1 response. Knockdown of MFN1 or MAVS can inhibit the HCMV-induced IFN1 production. What is more, confocal laser-scanning microscope showed that knockdown of MFN1 inhibits the HCMV-induced redistribution of MAVS. Conversely, MFN1 agonist leflunomide could induce IFN1 production. In conclusion, we provide new insight into the relationship between MFN1 and IFN1 during HCMV infection and show that MFN1 may serve as a potential strategy against HCMV infection.

Development and evaluation of machine learning models and nomogram for the prediction of severe acute pancreatitis.

BACKGROUND AND AIM: Severe acute pancreatitis (SAP) in patients progresses rapidly and can cause multiple organ failures associated with high mortality. We aimed to train a machine learning (ML) model and establish a nomogram that could identify SAP, early in the course of acute pancreatitis (AP). METHODS: In this retrospective study, 631 patients with AP were enrolled in the training cohort. For predicting SAP early, five supervised ML models were employed, such as random forest (RF), K-nearest neighbors (KNN), and naive Bayes (NB), which were evaluated by accuracy (ACC) and the areas under the receiver operating characteristic curve (AUC). The nomogram was established, and the predictive ability was assessed by the calibration curve and AUC. They were externally validated by an independent cohort of 109 patients with AP. RESULTS: In the training cohort, the AUC of RF, KNN, and NB models were 0.969, 0.954, and 0.951, respectively, while the AUC of the Bedside Index for Severity in Acute Pancreatitis (BISAP), Ranson and Glasgow scores were only 0.796, 0.847, and 0.837, respectively. In the validation cohort, the RF model also showed the highest AUC, which was 0.961. The AUC for the nomogram was 0.888 and 0.955 in the training and validation cohort, respectively. CONCLUSIONS: Our findings suggested that the RF model exhibited the best predictive performance, and the nomogram provided a visual scoring model for clinical practice. Our models may serve as practical tools for facilitating personalized treatment options and improving clinical outcomes through pre-treatment stratification of patients with AP.

Single-Cell Transcriptome Reveals the Metabolic and Clinical Features of a Highly Malignant Cell Subpopulation in Pancreatic Ductal Adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with a high mortality rate. PDAC exhibits significant heterogeneity as well as alterations in metabolic pathways that are associated with its malignant progression. In this study, we explored the metabolic and clinical features of a highly malignant subgroup of PDAC based on single-cell transcriptome technology. Methods: A highly malignant cell subpopulation was identified at single-cell resolution based on the expression of malignant genes. The metabolic landscape of different cell types was analyzed based on metabolic pathway gene sets. In vitro experiments to verify the biological functions of the marker genes were performed. PDAC patient subgroups with highly malignant cell subpopulations were distinguished according to five glycolytic marker genes. Five glycolytic highly malignant-related gene signatures were used to construct the glycolytic highly malignant-related genes signature (GHS) scores. Results: This study identified a highly malignant tumor cell subpopulation from the single-cell RNA sequencing (scRNA-seq) data. The analysis of the metabolic pathway revealed that highly malignant cells had an abnormally active metabolism, and enhanced glycolysis was a major metabolic feature. Five glycolytic marker genes that accounted for the highly malignant cell subpopulations were identified, namely, EN O 1, LDHA, PKM, PGK1, and PGM1. An in vitro cell experiment showed that proliferation rates of PANC-1 and CFPAC-1 cell lines decreased after knockdown of these five genes. Patients with metabolic profiles of highly malignant cell subpopulations exhibit clinical features of higher mortality, higher mutational burden, and immune deserts. The GHS score evaluated using the five marker genes was an independent prognostic factor for patients with PDAC. Conclusion: We revealed a subpopulation of highly malignant cells in PDAC with enhanced glycolysis as the main metabolic feature. We obtained five glycolytic marker gene signatures, which could be used to identify PDAC patient subgroups with highly malignant cell subpopulations, and proposed a GHS prognostic score.

Efficacy and Safety of Transcutaneous Electrical Acupoint Stimulation (TEAS) for Postoperative Pain in Laparoscopy: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

OBJECTIVES: This meta-analysis aimed to assess the efficacy and safety of transcutaneous acupoint electrical stimulation (TEAS) for postoperative pain in laparoscopy. The review has been registered on the "INPLASY" website and the registration number is INPLASY202150101. METHODS: Relevant randomized controlled trials are selected from seven electronic databases (PubMed, the Cochrane Library, Embase, China National Knowledge Infrastructure, Chongqing VIP Information, WanFang Data, and Chinese Biomedical Database) from their inception up to November 30, 2020. Twenty-eight studies were included in this meta-analysis, and the statistical analyses and the exploration of heterogeneity sources were conducted by Stata 15.0 software. Besides, the bias assessment of the included studies was evaluated using the Cochrane risk of bias tool. RESULTS: In total, 28 RCTs covering 2787 participants were included. The meta-analysis suggested that TEAS can effectively relieve pain in the short term after laparoscopy, reduce the postoperative consumption of rescue analgesics, improve the quality of life of patients, and shorten the length of hospitalization. And no serious adverse events are related to TEAS. Therefore, TEAS is relatively safe and efficacy for clinical application. The most used acupoints were Hegu (LI14), Neiguan (PC6), and Zusanli (ST36). CONCLUSIONS: TEAS can be recommended as a complementary and alternative therapy for the treatment of postoperative pain after laparoscopy. However, the included RCTs had some methodological limitations. Therefore, larger-size, more rigorous, and higher-quality RCTs are needed in the future to further explore the efficacy and safety of TEAS for postoperative pain after laparoscopy.

Efficacy and Safety of Banxia Formulae for Insomnia: A Systematic Review and Meta-Analysis of High-Quality Randomized Controlled Trials.

OBJECTIVE: To systematically evaluate the efficacy and safety of Banxia (Pinellia Tuber) formulae in the treatment of insomnia compared with those of conventional western medicines. METHODS: Randomized controlled trials (RCTs) evaluating the efficacy and safety of Banxia formulae in the treatment of insomnia were searched from the following databases: PubMed, Cochrane Library, EMBASE, the China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Database (VIP), and Wanfang database. The literature collected was from the time when the databases were established to April 2020. Quality assessment and meta-analysis were conducted by using Cochrane bias risk assessment tool and RevMan 5.2, respectively. Publication bias was assessed by Egger's test. RESULTS: Fourteen RCTs with 910 participants were identified. A total of 46 traditional Chinese medicines involving 2 different dosage forms were used in the included studies. Meta-analysis indicated that Banxia formulae had more significant effects on improving the total effective rate (RR = 1.23, 95% CI 1.16 to 1.31), Pittsburgh Sleep Quality Index (PSQI, MD = -1.05, 95% CI -1.63 to -0.47), and the TCM syndrome score (SMD = -0.78, 95% CI -1.18 to -0.39). Meanwhile, on reducing adverse events, Banxia formulae also showed an advantage (RR = 0.48, 95% CI 0.24 to 0.93). CONCLUSION: According to the current studies, the efficacy of Banxia formulae in the treatment of insomnia is better than that of the conventional western medicines, and its safety is relatively stable. However, due to the limitations of this study, further research and evaluation are needed.

Exploratory development of PCR-fluorescent probes in rapid detection of mutations associated with extensively drug-resistant tuberculosis.

This study aims to evaluate the clinical value of PCR-fluorescent probes for detecting the mutation gene associated with extensively drug-resistant tuberculosis (XDR-TB). The molecular species identification of 900 sputum specimens was performed using polymerase chain reaction (PCR)-fluorescent probe. The mutations of the drug resistance genes rpoB, katG, inhA, embB, rpsL, rrs, and gyrA were detected. The conventional drug susceptibility testing (DST) and PCR-directed sequencing (PCR-DS) were carried out as control. DST demonstrated that there were 501 strains of rifampicin resistance, 451 strains of isoniazid resistance, 293 strains of quinolone resistance, 425 strains of streptomycin resistance, 235 strains of ethambutol resistance, and 204 strains of amikacin resistance. Furthermore, 427 (47.44%) or 146 (16.22%) strains were MDR-TB or XDR-TB, respectively. The mutations of the rpoB, katG, inhA, embB, rpsL, rrs, and gyrA genes were detected in 751 of 900 TB patients by PCR-fluorescent probe method, and the rate of drug resistance was 751/900 (83.44%). No mutant genes were detected in the other 149 patients. Compared with DST, the mutant rates of rpoB, katG/inhA, rpsL, rrs, embB, and gyrA of six drugs were higher than 88%; five of six drugs were higher than 90% except for SM (88.11%). The MDR and XDR mutant gene types were found in 398 (42.22%) and 137 (15.22%) samples. PCR-DS was also employed and confirmed the PCR-fluorescent probe method with the accordance rate of 100%. The PCR-fluorescent probe method is rapid and straightforward in detecting XDR-TB genotypes and is worthy of being applied in hospitals.

Rapid and visual detection of Mycobacterium tuberculosis complex using recombinase polymerase amplification combined with lateral flow strips.

To definitively diagnose active pulmonary Tuberculosis (TB), Mycobacterium tuberculosis complex (MTBC) bacilli must be identified within clinical specimens from patients. In this study, we introduced a rapid and visual detection method of MTBC using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strips. The LF-RPA assay, read results with naked eyes, could detect as few as 5 genome copies of M. tuberculosis H37Rv (ATCC 27294) per reaction and had no cross-reactions with other control bacteria even using excessive amount of template DNA. The system could work well at a broad range of temperature 25-45 °C and reach detectable level even within 5 min. When testing a total of 137 clinical specimens, the sensitivity and specificity of the LF-RPA assay were 100% (95% CI: 95.94%-100%) and 97.92% (95% CI: 88.93%-99.95%), respectively, compared to culture identification method. Therefore, the LF-RPA system we have demonstrated is a rapid, simple, robust method for MTBC detection which, subject to the availability of a suitable sample extraction method, has the potentiality to diagnose TB at the point-of-care testing.

A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia.

An air-insulated microfluidic chip was designed for the automatic centrifugal distribution of samples to 24-test cells, enabling the parallel identification of multiple clinical pneumonia-related pathogens in 1.45-µL reactions without cross-contamination in 45 min. A portable nucleic acid analyzer that integrates mechanical, confocal optical, electronic, and software functions was also developed to collect fluorescence data in a Ø3 mm imaging field near the optical diffraction limit for highly sensitive fluorescence detection of nucleic acid amplification in real time. This microfluidic chip-based portable nucleic acid analyzer could detect low abundance nucleic acids present at as few as 10 copies. In a blinded experiment, specific identification of Mycoplasma pneumoniae, Staphylococcus aureus, and methicillin-resistant S. aureus was achieved with 229 clinical patient sputum samples. The total coincidence rate of our system and traditional RT-PCR with an ABI 7500 was 99.56%. Four samples accounting for the 0.44% inconformity were retested by gene sequencing, revealing that our system reported the correct results. This novel microfluidic chip-based detection system is cost-effective, rapid, sensitive, specific, and has a relatively high throughput for parallel identification, which is especially suitable for resource-limited facilities/areas and point-of-care testing.

Screening of deafness-causing DNA variants that are common in patients of European ancestry using a microarray-based approach.

The unparalleled heterogeneity in genetic causes of hearing loss along with remarkable differences in prevalence of causative variants among ethnic groups makes single gene tests technically inefficient. Although hundreds of genes have been reported to be associated with nonsyndromic hearing loss (NSHL), GJB2, GJB6, SLC26A4, and mitochondrial (mt) MT-RNR1 and MTTS are the major contributors. In order to provide a faster, more comprehensive and cost effective assay, we constructed a DNA fluidic array, CapitalBioMiamiOtoArray, for the detection of sequence variants in five genes that are common in most populations of European descent. They consist of c.35delG, p.W44C, p.L90P, c.167delT (GJB2); 309kb deletion (GJB6); p.L236P, p.T416P (SLC26A4); and m.1555A>G, m.7444G>A (mtDNA). We have validated our hearing loss array by analyzing a total of 160 DNAs samples. Our results show 100% concordance between the fluidic array biochip-based approach and the established Sanger sequencing method, thus proving its robustness and reliability at a relatively low cost.

Association between glycated hemoglobin A1c levels with age and gender in Chinese adults with no prior diagnosis of diabetes mellitus.

The present cross-sectional study consisted of 18,265 Chinese patients not previously diagnosed with diabetes mellitus, and who underwent physical examination at the Third People's Hospital of Shenzhen between June 2014 and May 2015 (mean patient age, 51.312±15.252 years). The study was composed of 11,770 males and 6,495 females. The aim was to investigate the association between glycated hemoglobin A1c (HbA1c) levels, gender and age. HbA1c values were measured using a Bio-Rad VARIANT™ II HbA1c Reorder Pack. All data was collected for analysis of the HbA1c levels in different gender and age groups, in order to investigate the association between HbA1c levels and age. Analysis of the 18,265 total cases and 16,734 cases with HbA1c levels <6.5%, demonstrated a positive correlation between levels of HbA1c and patient age. Linear regression for patient age and HbA1c levels demonstrated that HbA1c (%) = 0.020 × age (years) + 4.523 (r=0.369, P<0.0001) and HbA1c (%) = 0.014 × age (years) + 4.659 (r=0.485, P<0.0001), respectively. HbA1c levels of the male group were significantly higher than those of the female group (P<0.0001). Furthermore, in different gender groups, HbA1c levels gradually rose with increasing age. Therefore, HbA1c levels are associated with age and gender in Chinese populations, and this should be considered when selecting HbA1c as a criterion for future diabetes screening.

A fully integrated and automated microsystem for rapid pharmacogenetic typing of multiple warfarin-related single-nucleotide polymorphisms.

A fully integrated and automated microsystem consisting of low-cost, disposable plastic chips for DNA extraction and PCR amplification combined with a reusable glass capillary array electrophoresis chip in a modular-based format was successfully developed for warfarin pharmacogenetic testing. DNA extraction was performed by adopting a filter paper-based method, followed by "in situ" PCR that was carried out directly in the same reaction chamber of the chip without elution. PCR products were then co-injected with sizing standards into separation channels for detection using a novel injection electrode. The entire process was automatically conducted on a custom-made compact control and detection instrument. The limit of detection of the microsystem for the singleplex amplification of amelogenin was determined to be 0.625 ng of standard K562 DNA and 0.3 µL of human whole blood. A two-color multiplex allele-specific PCR assay for detecting the warfarin-related single-nucleotide polymorphisms (SNPs) 6853 (-1639G>A) and 6484 (1173C>T) in the VKORC1 gene and the *3 SNP (1075A>C) in the CYP2C9 gene was developed and used for validation studies. The fully automated genetic analysis was completed in two hours with a minimum requirement of 0.5 µL of input blood. Samples from patients with different genotypes were all accurately analyzed. In addition, both dried bloodstains and oral swabs were successfully processed by the microsystem with a simple modification to the DNA extraction and amplification chip. The successful development and operation of this microsystem establish the feasibility of rapid warfarin pharmacogenetic testing in routine clinical practice.

Mutation screening for thalassaemia in the Jino ethnic minority population of Yunnan Province, Southwest China.

OBJECTIVES: This study aimed to detect α- and ß-thalassaemia mutations in the Jino ethnic minority population of Yunnan Province, Southwest China. DESIGN: A total of 1613 Jino adults were continuously recruited from February 2012 to April 2012. Fasting venous blood samples were obtained to determine haematological variables. Haemoglobin analysis was conducted using high-performance liquid chromatography. Participants with hypochromic microcytic anaemia or positive haemoglobin analysis profiles were confirmed by α- and ß-globin genetic testing, including DNA microarray analysis, direct sequencing methods and multiplex gap-PCR assays. SETTING: Shanghai Diabetes Institute, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Jiao Tong University Affiliated Sixth People's Hospital. RESULTS: We found 363 suspected cases by primary screening of haematological variables and haemoglobin analysis. After further genetic testing, four types of α- and ß-thalassaemia mutation were detected in 203 out of 363 individuals. Both α(0)- and α(+)-thalassaemia mutations, --(SEA) and -α(3.7), were identified. ß-Thalassaemia mutations included CD17 (HBB:c.52A>T) and CD26 (HbE or HBB:c.79G>A). In addition, 13 HbE carriers had coexisting α(0)- or α(+)-thalassaemia deletions. Clinical haematological variables indicated that, in this study, carriers of all thalassaemic genotypes had more severe hypochromic microcytic anaemia than non-thalassaemic individuals. CONCLUSIONS: Our results provide information on the Jino ethnic minority that may be useful for further genetic counselling, prenatal screening and clinical diagnosis of thalassaemia in this region.

Microarray-based genotyping and detection of drug-resistant HBV mutations from 620 Chinese patients with chronic HBV infection

Background: Research has shown that hepatitis B virus (HBV) genotypes are closely linked to the clinical manifestations, treatment, and prognosis of the disease. Objective: To study the association between genotype and drug-resistant HBV mutations in 620 Chinese patients with chronic HBV infection. Methods: HBV DNA levels were determined using real-time quantitative PCR in plasma samples. Microarrays were performed for the simultaneous detection of HBV genotypes (HBV/B, C, and D) and drug-resistance-related hotspot mutations. A portion of the samples analyzed using microarrays was selected randomly and the data were confirmed using direct DNA sequencing. Results: Most samples were genotype C (471/620; 76.0%), followed by genotype B (149/620; 24.0%). Among the 620 patient samples, 17 (2.7%) had nucleotide analogs (NA) resistance-related mutations. Of these, nine and eight patients carried lamivudine (LAM)-/telbivudine (LdT)-resistance mutations (rtL180M, rtM204I/V) and adefovir (ADV)-resistance mutations (rtA181T/V, rtN236T), respectively. No patients had both lamivudine (LAM)- and either ade-fovir (ADV) or entecavir (ETV) resistance mutations. Additionally, out of the 620 patient samples, 64.0% (397/620) were also detected with the precore stop-codon mutation (G1896A) by microarray assay. Conclusion: The results of the current study revealed that the prevalence of nucleotide analogs (NA)-resistance in Chinese hospitalized HBV-positive patients was so low that intensive nucleotide analogs (NA)-resistance testing before nucleotide analog (NA) treatment might not be required. In addition, the present study suggests that chronic HBV patients with genotype C were infected with fitter viruses and had an increased prevalence of nucleotide analogs (NA)-resistance mutations compared to genotype B virus. .

Microarray-based genotyping and detection of drug-resistant HBV mutations from 620 Chinese patients with chronic HBV infection.

BACKGROUND: Research has shown that hepatitis B virus (HBV) genotypes are closely linked to the clinical manifestations, treatment, and prognosis of the disease. OBJECTIVE: To study the association between genotype and drug-resistant HBV mutations in 620 Chinese patients with chronic HBV infection. METHODS: HBV DNA levels were determined using real-time quantitative PCR in plasma samples. Microarrays were performed for the simultaneous detection of HBV genotypes (HBV/B, C, and D) and drug-resistance-related hotspot mutations. A portion of the samples analyzed using microarrays was selected randomly and the data were confirmed using direct DNA sequencing. RESULTS: Most samples were genotype C (471/620; 76.0%), followed by genotype B (149/620; 24.0%). Among the 620 patient samples, 17 (2.7%) had nucleotide analogs (NA) resistance-related mutations. Of these, nine and eight patients carried lamivudine (LAM)-/telbivudine (LdT)-resistance mutations (rtL180M, rtM204I/V) and adefovir (ADV)-resistance mutations (rtA181T/V, rtN236T), respectively. No patients had both lamivudine (LAM)- and either adefovir (ADV) or entecavir (ETV) resistance mutations. Additionally, out of the 620 patient samples, 64.0% (397/620) were also detected with the precore stop-codon mutation (G1896A) by microarray assay. CONCLUSION: The results of the current study revealed that the prevalence of nucleotide analogs (NA)-resistance in Chinese hospitalized HBV-positive patients was so low that intensive nucleotide analogs (NA)-resistance testing before nucleotide analog (NA) treatment might not be required. In addition, the present study suggests that chronic HBV patients with genotype C were infected with fitter viruses and had an increased prevalence of nucleotide analogs (NA)-resistance mutations compared to genotype B virus.

Rapid identification of aminoglycoside-induced deafness gene mutations using multiplex real-time polymerase chain reaction.

BACKGROUND: Exposure to aminoglycoside antibiotics can induce ototoxicity in genetically susceptible individuals carrying certain mitochondrial DNA (mtDNA) mutations (C1494T and A1555G), resulting in hearing loss. So, a rapid diagnostic approach is needed to accurately identify subjects carrying such gene mutations. METHODS: In the present study, we describe a rapid and reliable four-color, real-time quantitative polymerase chain reaction (qPCR) assay for simultaneously detecting two mtDNA 12S rRNA gene variants, A1555G and C1494T, which are prevalent in the Han Chinese population. This multiplex assay incorporates three allele-specific TaqMan probes labeled with different fluorophores in a single reaction, providing high genotyping accuracy for clinical blood samples. RESULTS: Tests with C1494T, A1555G and wild-type DNA exhibited high sensitivity, specificity, reproducibility and accuracy of discriminating mutations from wild-type. CONCLUSIONS: This study shows that this simple and inexpensive method can be used for routine molecular diagnostics and potentially for large-scale genetic screening.

An automated microfluidic system for single-stranded DNA preparation and magnetic bead-based microarray analysis.

We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-µl pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, "amplicon-in-answer-out" operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis.

Fully automated sample preparation microsystem for genetic testing of hereditary hearing loss using two-color multiplex allele-specific PCR.

A fully automated microsystem consisting of a disposable DNA extraction and PCR microchip, as well as a compact control instrument, has been successfully developed for genetic testing of hereditary hearing loss from human whole blood. DNA extraction and PCR were integrated into a single 15-µL reaction chamber, where a piece of filter paper was embedded for capturing genomic DNA, followed by in-situ PCR amplification without elution. Diaphragm microvalves actuated by external solenoids together with a "one-way" fluidic control strategy operated by a modular valve positioner and a syringe pump were employed to control the fluids and to seal the chamber during thermal cycling. Fully automated DNA extractions from as low as 0.3-µL human whole blood followed by amplifications of 59-bp ß-actin fragments can be completed on the microsystem in about 100 min. Negative control tests that were performed between blood sample analyses proved the successful elimination of any contamination or carryover in the system. To more critically test the microsystem, a two-color multiplex allele-specific PCR (ASPCR) assay for detecting c.176_191del16, c.235delC, and c.299_300delAT mutations in GJB2 gene that accounts for hereditary hearing loss was constructed. Two allele-specific primers, one labeled with TAMRA for wild type and the other with FAM for mutation, were designed for each locus. DNA extraction from blood and ASPCR were performed on the microsystem, followed by an electrophoretic analysis on a portable microchip capillary electrophoresis system. Blood samples from a healthy donor and five persons with genetic mutations were all accurately analyzed with only two steps in less than 2 h.

A rapid automatic processing platform for bead label-assisted microarray analysis: application for genetic hearing-loss mutation detection.

Molecular diagnostics using microarrays are increasingly being used in clinical diagnosis because of their high throughput, sensitivity, and accuracy. However, standard microarray processing takes several hours and involves manual steps during hybridization, slide clean up, and imaging. Here we describe the development of an integrated platform that automates these individual steps as well as significantly shortens the processing time and improves reproducibility. The platform integrates such key elements as a microfluidic chip, flow control system, temperature control system, imaging system, and automated analysis of clinical results. Bead labeling of microarray signals required a simple imaging system and allowed continuous monitoring of the microarray processing. To demonstrate utility, the automated platform was used to genotype hereditary hearing-loss gene mutations. Compared with conventional microarray processing procedures, the platform increases the efficiency and reproducibility of hybridization, speeding microarray processing through to result analysis. The platform also continuously monitors the microarray signals, which can be used to facilitate optimization of microarray processing conditions. In addition, the modular design of the platform lends itself to development of simultaneous processing of multiple microfluidic chips. We believe the novel features of the platform will benefit its use in clinical settings in which fast, low-complexity molecular genetic testing is required.