Accurate quantification of DNA using on-site PCR (osPCR) by characterizing DNA amplification at single-molecule resolution.

Despite the need in various applications, accurate quantification of nucleic acids still remains a challenge. The widely-used qPCR has reduced accuracy at ultralow template concentration and is susceptible to nonspecific amplifications. The more recently developed dPCR is costly and cannot handle high-concentration samples. We combine the strengths of qPCR and dPCR by performing PCR in silicon-based microfluidic chips and demonstrate high quantification accuracy in a large concentration range. Importantly, at low template concentration, we observe on-site PCR (osPCR), where only certain sites of the channel show amplification. The sites have almost identical ct values, showing osPCR is a quasi-single molecule phenomenon. Using osPCR, we can measure both the ct values and the absolute concentration of templates in the same reaction. Additionally, osPCR enables identification of each template molecule, allowing removal of nonspecific amplification during quantification and greatly improving quantification accuracy. We develop sectioning algorithm that improves the signal amplitude and demonstrate improved detection of COVID in patient samples.

COVID-19-another influential event impacts on laboratory medicine management.

BACKGROUND: Before public health emergencies became a major challenge worldwide, the scope of laboratory management was only related to developing, maintaining, improving, and sustaining the quality of accurate laboratory results for improved clinical outcomes. Indeed, quality management is an especially important aspect and has achieved great milestones during the development of clinical laboratories. CURRENT STATUS: However, since the coronavirus disease 2019 (COVID-19) pandemic continues to be a threat worldwide, previous management mode inside the separate laboratory could not cater to the demand of the COVID-19 public health emergency. Among emerging new issues, the prominent challenges during the period of COVID-19 pandemic are rapid-launched laboratory-developed tests (LDTs) for urgent clinical application, rapid expansion of testing capabilities, laboratory medicine resources, and personnel shortages. These related issues are now impacting on clinical laboratory and need to be effectively addressed. CONCLUSION: Different from traditional views of laboratory medicine management that focus on separate laboratories, present clinical laboratory management must be multidimensional mode which should consider consolidation of the efficient network of regional clinical laboratories and reasonable planning of laboratories resources from the view of overall strategy. Based on relevant research and our experience, in this review, we retrospect the history trajectory of laboratory medicine management, and also, we provide existing and other feasible recommended management strategies for laboratory medicine in future.

External quality assessment for human papillomavirus 16/18 DNA detection and genotyping in Shanghai, China.

BACKGROUND: Human papillomavirus (HPV) DNA detection and genotyping is now being used for cervical screening by a growing number of laboratories in Shanghai, but they may have various levels of proficiency. The objective of this study was to evaluate the performance of clinical laboratories for HPV DNA detection and genotyping by an external quality assessment (EQA) program. METHODS: The EQA panels were clinically validated by the Cobas 4800 HPV test, and then distributed to the participating laboratories in May 2015 (round 1) and September 2015 (round 2). Each panel consisted of one negative sample and nine positive cell or clinical samples of HPV16 and HPV18 types at different concentrations. In total, 40 laboratories submitted 18 qualitative and 22 genotyping data sets in round 1 and 44 laboratories submitted 18 qualitative and 26 genotyping data sets in round 2. In both rounds, all laboratories used commercial assays. RESULTS: The negative samples were detected correctly in both rounds by all participating laboratories. There were no false-positive results in the qualitative data sets and only two false-positive results in the genotyping data sets in each of round 1 and round 2. The false-negative rates were 8.0% for round 1 and 2.7% for round 2. For the qualitative data sets, almost all of the laboratories (100% for round 1 and 97.8% for round 2) obtained a score of acceptable or better. For the genotyping results, acceptable or better scores were obtained in 81.8% (round 1) and 100% (round 2). CONCLUSIONS: Our results indicate that the majority of laboratories in Shanghai have reliable diagnostic ability for HPV detection and genotyping. Moreover, this study emphasizes the importance of EQA for monitoring the performance of clinical laboratories.

Use of chimeric influenza viruses as a novel internal control for diagnostic rRT-PCR assays.

Real-time quantitative reverse transcriptase polymerase chain reaction (rRT-PCR) is now widely used to detect viral pathogens in various human specimens. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by unexpected inhibition or inefficient extraction. In the present study, we describe a strategy to produce a stable internal control for rRT-PCR by packaging foreign RNA into influenza virions using plasmid-based reverse genetics technology. The envelope structure of influenza virus can effectively protect RNA segments from RNase digestion, which provides an advantage for its routine use as an internal control. Utilizing this approach, we successfully generated a recombinant influenza virus (rPR8-HCV) containing the 5' untranslated region (5'UTR) of the hepatitis C virus (HCV) RNA genome. After inactivation and purification, the rPR8-HCV particles were demonstrated to be RNase resistant and stable at 4 °C for at least 252 days in human plasma, with no degradation even after being frozen and thawed multiple times. These results were reproducible in the COBAS TaqMan HCV test for 164 days. Moreover, the chimeric influenza virus particles could be easily produced in embryonated eggs and were noninfectious after inactivation treatment. Additionally, this strategy could also be adapted for real-time clinical applications of other RNA targets, providing a universal approach with broad clinical applications in rRT-PCR assays.

Construction and application of a heterogeneous quality control library for the Xpert MTB/RIF assay in tuberculosis diagnosis.

Proficiency testing based on quality control materials is an important component of the quality assurance system for detection methods. However, in the detection of infectious diseases, it is a challenge to use quality control materials derived from clinical samples or pathogens owing to their infectious nature. The Xpert MTB/RIF assay, endorsed by the World Health Organization, is one of the most widely implemented assays in the detection of Mycobacterium tuberculosis along with rifampicin resistance and its heterogeneity. Clinical isolates are typically used as quality controls for this assay, leading to concerns about biosafety, constrained target sequence polymorphisms, and time-consuming preparation. In this study, a heterogeneous quality control library for the Xpert MTB/RIF assay was constructed based on DNA synthesis and site-directed mutation, which provides sufficient rifampicin resistance polymorphisms, enabling monitoring all five probes of Xpert MTB/RIF and its combinations. Escherichia coli and Bacillus subtilis were used as heterogeneous hosts rather than the pathogen itself to eliminate biosafety risks; thus, preparation does not require a biosafety level III laboratory and the production time is reduced from a few months to a few days. The panel was stable for more than 15 months stored at 4°C and could be distributed at room temperature. All 11 laboratories in Shanghai participating in a pilot survey identified the specimens with corresponding probe patterns, and discordant results highlighted inappropriate operations in the process. Collectively, we show, for the first time, that this library, based on heterogeneous hosts, is an appropriate alternative for M. tuberculosis detection.

Design scheme of a high dynamic semi-airborne transient electromagnetic receiving system based on a voltage-controlled amplifier.

The semi-airborne transient electromagnetic (SATEM) technique is established to examine subsurface targets. The SATEM approach is widely used in urban underground space exploration, hydrogeological search, and deep mineral exploration. However, the strength of SATEM signals declines exponentially with the depth of detection, and the SATEM system frequently operates in high electromagnetic noise environments, directly affecting the precision of deep information interpretation. This research presents a high dynamic semi-airborne transient electromagnetic (HDSATEM) receiving system to increase the late SATEM signal intensity. This system mainly comprises a voltage-controlled amplifier (VCA) and a function generator. In addition, the VCA is modulated using the gain voltage produced by the function generator. Consequently, a simulation model has been employed to analyze various functions to compare the dynamic ranges. The actual amplification performance and signal restoration capacity are confirmed using a laboratory test. A field experiment is also carried out to evaluate the actual detection performance of the proposed system compared to standard SATEM receiving and controlled source audio-frequency magneto-telluric systems. The findings indicate that the performance improvement of the HDSATEM receiving system is more than 50% in terms of depth detection compared to that of standard SATEM receiving systems.

Fibrinogen BOE II: dysfibrinogenemia with bleeding and defective thrombin binding.

Background: Variants of fibrinogen sequences that bind to thrombin's catalytic sites are mostly associated with bleeding phenotypes, while variants with fibrinogen nonsubstrate-thrombin-binding sites are commonly believed to cause thrombosis. AαGlu39 and BßAla68 play important roles in fibrin(ogen)-thrombin-nonsubstrate binding. The BßAla68Thr variant has been described in several unrelated families with apparent thrombotic phenotypes. Objectives: Homozygous AαGlu39Lys variant (fibrinogen BOE II) was identified in a boy with dysfibrinogenemia who had multiple cerebral hemorrhages. A series of analyses were performed to assess the variant's functions and elucidate underlying bleeding mechanisms. Methods: Abnormal fibrinogen was purified from plasma and subjected to Western blot, fibrinogen and fibrin monomer polymerization, clottability, fibrinopeptides release, activated factor (F)XIII (FXIIIa) cross-linking, fibrinolysis, and scanning electron microscopy analyses. Results: Fibrinogen BOE II weakened the binding capacity of thrombin to fibrinogen and delayed the formation of fibrin clots. The release of fibrinopeptides, polymerization of fibrinogen catalyzed by thrombin, and cross-linking of FXIIIa of fibrinogen BOE II were impaired. In contrast, batroxobin-catalyzed fibrinogen polymerization and desA/desAB fibrin monomer polymerization did not differ from those in normal controls. Fibrin clots formed by fibrinogen BOE II were composed of thicker fibrin fibers and showed a faster fibrinolysis rate. Conclusion: Defective fibrin(ogen)-thrombin-nonsubstrate binding is not necessarily associated with thrombotic disorders. When the hypercoagulable state created by increased circulating free thrombin is insufficient to compensate for defective hemostasis caused by slowly formed but rapidly lysed clots, the primary concern of thrombin-binding deficiency dysfibrinogenemia appears to be hemorrhage rather than thrombosis.

Hierarchical Self-Supervised Learning for 3D Tooth Segmentation in Intra-Oral Mesh Scans.

Accurately delineating individual teeth and the gingiva in the three-dimension (3D) intraoral scanned (IOS) mesh data plays a pivotal role in many digital dental applications, e.g., orthodontics. Recent research shows that deep learning based methods can achieve promising results for 3D tooth segmentation, however, most of them rely on high-quality labeled dataset which is usually of small scales as annotating IOS meshes requires intensive human efforts. In this paper, we propose a novel self-supervised learning framework, named STSNet, to boost the performance of 3D tooth segmentation leveraging on large-scale unlabeled IOS data. The framework follows two-stage training, i.e., pre-training and fine-tuning. In pre-training, three hierarchical-level, i.e., point-level, region-level, cross-level, contrastive losses are proposed for unsupervised representation learning on a set of predefined matched points from different augmented views. The pretrained segmentation backbone is further fine-tuned in a supervised manner with a small number of labeled IOS meshes. With the same amount of annotated samples, our method can achieve an mIoU of 89.88%, significantly outperforming the supervised counterparts. The performance gain becomes more remarkable when only a small amount of labeled samples are available. Furthermore, STSNet can achieve better performance with only 40% of the annotated samples as compared to the fully supervised baselines. To the best of our knowledge, we present the first attempt of unsupervised pre-training for 3D tooth segmentation, demonstrating its strong potential in reducing human efforts for annotation and verification.

Analytical sensitivity comparison of 14 conventional and three rapid RT-PCR assays for SARS-CoV-2 detection.

Recent reports have compared the analytical sensitivities of some SARS-CoV-2 RT-PCR assays, but differences in the viral materials used for these evaluations made comprehensive conclusions difficult. We carried out a direct comparison of the analytical sensitivities of 14 conventional and three rapid RT-PCR assays for the detection of SARS-CoV-2. The comparison was performed utilizing a certified reference material for SARS-CoV-2 RNA that was serially two-fold diluted in RNA storage solution. Our results show that the analytical sensitivities of the 17 assays varied within an 8-fold range (100-800 copies/mL). Moreover, a trend with some rapid assays yielding slightly higher analytical sensitivities (2- to 4-fold) compared with conventional assays was observed. We conclude that most of the RT-PCR assays can be used for routine COVID-19 diagnosis, but some assays with the poorest analytical sensitivities may lead to false-negative results when used to identify asymptomatic individuals who can carry a low viral load but still be infectious. These findings should be kept in mind when selecting high-sensitivity and rapid assays.

Evaluation of three sample preparation methods for the identification of clinical strains by using two MALDI-TOF MS systems.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the microbial identification, especially in the clinical microbiology laboratories. However, although numerous studies on the identification of microorganisms by MALDI-TOF MS have been reported previously, few studies focused on the effect of pretreatment on identification. Due to the sensitivity of MALDI-TOF MS, different preparation methods will lead to changes in microbial protein fingerprints. In this study, for evaluating a more appropriate preparation method for the clinical microbiology identification, we analyzed the performance of three sample preparation methods on two different MALDI-TOF MS systems. A total of 321 clinical isolates, 127 species, were employed in the comparative study of three different sample preparation methods including the direct colony transfer method (DCTM), the on-target extraction method (OTEM), and the in-tube extraction method (ITEM) compatible with MALDI-TOF MS. All isolates were tested on the Microflex LT and Autof ms1000 devices. The spectra were analyzed using the Bruker biotyper and the Autof ms1000 systems. The results were confirmed by 16/18S rRNA sequencing. Results reveal that the accuracies of isolates identification by Bruker biotyper successfully identified 83.8%, 96.0%, and 95.3% after performing the DCTM, OTEM, and ITEM, respectively, while the Autof ms1000 identified 97.5%, 100%, and 99.7%. These data suggested that the identification rates are comparable among the three preparation methods using the Autof ms1000 and Bruker microflex LT systems but the OTEM is more suitable and necessary for clinical application, owing to its key advantages of simplicity and accuracy.

Up-regulated long noncoding RNA AC007128.1 and its genetic polymorphisms associated with Tuberculosis susceptibility.

BACKGROUND: Tuberculosis (TB) remains a major public health problem. Long non-coding RNAs (lncRNAs) are important regulators of gene expression. In this study, we explored the association between the expression of lncRNA AC007128.1 and TB susceptibility. METHODS: Three single-nucleotide polymorphisms (SNPs) (rs12333784, rs6463794, and rs720964) of lncRNA AC007128.1 were selected using the 1000 Genomes Project database and offline software Haploview V4.2, and were genotyped by a customized 2×48-Plex SNPscan™ Kit. RESULTS: We identified two differentially expressed lncRNA including AC007128.1 and AP001065.3 in comparisons of expression profiles between ATB vs. LTBI, LTBI vs. HCs, and AC700128.1 expression was specifically and significantly up-regulated in TB patients by verification of external data. Gene Ontology functional enrichment analysis and co-expression network showed up-regulated mRNA was mainly involved in negative regulation of the G protein-coupled receptor (GPCR) signaling pathway, and FPR1 and CYP27B1 were involved in the co-expression of AC007128.1. Using the 1000 Genomes Project, software Haploview V4.2, and SNP genotype, we screened out SNP rs12333784 which locus at 7p21.3 in AC007128.1 associated with TB susceptibility. The G carrier of rs12333784 was then finally verified to be significantly associated with pulmonary TB (PTB) and extrapulmonary tuberculosis (EPTB) susceptibility (pBonferroni =0.03878), and a similar but more significant effect was observed under the dominant model analysis (pBonferroni =0.013, OR =1.349, 95% CI, 1.065-1.709). In addition, the GG + GA genotype of SNP rs12333784 was significantly correlated with higher glucose (GLU) (P=0.03), higher gamma-glutamyl transferase (GGT) (P=0.05), and higher erythrocyte sedimentation rate (ESR) (P=0.05). CONCLUSIONS: Our findings show lncRNA AC007128.1 can be regarded as biomarkers discriminating between ATB and LTBI and may also be a diagnostic biomarker for LBTI. These findings may aid clinical decision making in the management of TB.

Identification of antibiotic resistance genes in the multidrug-resistant Acinetobacter baumannii strain, MDR-SHH02, using whole-genome sequencing.

This study aimed to investigate antibiotic resistance genes in the multidrug-resistant (MDR) Acinetobacter baumannii (A. baumanii) strain, MDR-SHH02, using whole­genome sequencing (WGS). The antibiotic resistance of MDR-SHH02 isolated from a patient with breast cancer to 19 types of antibiotics was determined using the Kirby­Bauer method. WGS of MDR-SHH02 was then performed. Following quality control and transcriptome assembly, functional annotation of genes was conducted, and the phylogenetic tree of MDR-SHH02, along with another 5 A. baumanii species and 2 Acinetobacter species, was constructed using PHYLIP 3.695 and FigTree v1.4.2. Furthermore, pathogenicity islands (PAIs) were predicted by the pathogenicity island database. Potential antibiotic resistance genes in MDR-SHH02 were predicted based on the information in the Antibiotic Resistance Genes Database (ARDB). MDR-SHH02 was found to be resistant to all of the tested antibiotics. The total draft genome length of MDR-SHH02 was 4,003,808 bp. There were 74.25% of coding sequences to be annotated into 21 of the Clusters of Orthologous Groups (COGs) of protein terms, such as 'transcription' and 'amino acid transport and metabolism'. Furthermore, there were 45 PAIs homologous to the sequence MDRSHH02000806. Additionally, a total of 12 gene sequences in MDR-SHH02 were highly similar to the sequences of antibiotic resistance genes in ARDB, including genes encoding aminoglycoside­modifying enzymes [e.g., aac(3)-Ia, ant(2'')­Ia, aph33ib and aph(3')-Ia], ß-lactamase genes (bl2b_tem and bl2b_tem1), sulfonamide-resistant dihydropteroate synthase genes (sul1 and sul2), catb3 and tetb. These results suggest that numerous genes mediate resistance to various antibiotics in MDR-SHH02, and provide a clinical guidance for the personalized therapy of A. baumannii-infected patients.

Study on atherosclerosis treated with theory of detoxification.

Starting with the contents, classification and pathogenic characteristics of the toxic pathogen and combining the modern medical research on the correlation of atherosclerosis with inflammation and immune reaction, authors have studied and expounded the interrelationship between the toxic pathogen and atherosclerosis. The toxic pathogen affecting the whole pathological process of atherosclerosis is a key factor for the disease to remain lingering and a cause of various cardiocerebrovascular diseases. Detoxification can be used to treat atherosclerosis so as to enhance the toxin-removing ability of the body and resist the damage to the body from the toxic pathogen.

Dual Physically Cross-Linked Double Network Hydrogels with High Mechanical Strength, Fatigue Resistance, Notch-Insensitivity, and Self-Healing Properties.

Double-network (DN) hydrogels with high strength and toughness have been developed as promising materials. Herein, we explored a dual physically cross-linked polyacrylamide/xanthan gum (PAM/XG) DN hydrogel. The nonchemically cross-linked PAM/XG DN hydrogels exhibited fracture stresses as high as 3.64 MPa (13 times higher than the pure PAM single network hydrogel) and compressive stresses at 99% strain of more than 50 MPa. The hydrogels could restore their original shapes after continuously loading-unloading tensile and compressive cyclic tests. In addition, the PAM/XG DN hydrogels demonstrated excellent fatigue resistance, notch-insensitivity, high stability in different harsh environments, and remarkable self-healing properties, which might result from their distinctive physical-cross-linking structures. The attenuated total reflectance infrared spectroscopy (ATR-IR) and dynamic thermogravimetric analysis (TGA) results indicated that there were no chemical bonds (only hydrogen bonds) between the XG and PAM networks. The PAM/XG DN hydrogel synthesis offers a new avenue for the design and construction of DN systems, broadening current research and applications of hydrogels with excellent mechanical properties.

Detection of autoantibodies in the serum of primary hepatocarcinoma patients.

OBJECTIVE: To study the significance of detecting autoantibodies in primary hepatocarcinoma (PHC) patients. METHODS: Autoantibodies were detected by indirect immunofluorescence assay. Antigens and antibodies of HBV were determined by enzyme immune assay. Antibody to HCV IgG was detected by enzyme-linked immunoabsorbent assay. RESULTS: The positive rate of autoantibody was 27.3% (38/139) in 139 PHC patients. The main type of autoantibodies in PHC was anti-nuclear antibody (36/38, 94.7%), others included anti-smooth muscle antibody(2/38, 5.3%), anti-mitochondria antibody (1/38, 2.6%), anti-midbody antibody (1/38, 2.6%, and anti-liver cell membrane antibody (2/38, 5.3%). CONCLUSIONS: Detecting autoantibodies in PHC patients is of significance in studying the mechanism of autoimmune reaction and etiology in PHC. The diversity of autoantibodies might result from a wide variety of etiological factors involved in PHC development, and from a wide variety of overexpressed or mutated proteins involved in repeated cycles of necrosis and regeneration in hepatocarcinoma development.