Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Multiplexing has been highlighted to save on costs, increase sample throughput, and maximize on the number of targets that can be sensitively detected within a small sample. With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. In this study, we developed simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and fourplex (4 targets) assays based on a two color ddPCR system for SARS-CoV-2 detection. Results showed that the fourplex assay had the similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical isolates demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug couldnt. Conclusively, our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.

Integrated characterization of SARS-CoV-2 genome, microbiome, antibiotic resistance and host response from single throat swabs

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, poses a severe threat to humanity. Rapid and comprehensive analysis of both pathogen and host sequencing data is critical to track infection and inform therapies. In this study, we performed unbiased metatranscriptomic analysis of clinical samples from COVID-19 patients using a newly-developed RNA-seq library construction method (TRACE-seq), which utilizes tagmentation activity of Tn5 on RNA/DNA hybrids. This approach avoids the laborious and time-consuming steps in traditional RNA-seq procedure, and hence is fast, sensitive and convenient. We demonstrated that TRACE-seq allowed integrated characterization of full genome information of SARS-CoV-2, putative pathogens causing coinfection, antibiotic resistance and host response from single throat swabs. We believe that the integrated information will deepen our understanding of pathogenesis and improve diagnostic accuracy for infectious diseases.

Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP

The ability to detect an infectious agent in a widespread epidemic is crucial to the success of quarantine efforts in addition to sensitive and accurate screening of potential cases of infection from patients in a clinical setting. Enabling testing outside of sophisticated laboratories broadens the scope of control and surveillance efforts, but also requires robust and simple methods that can be used without expensive instrumentation. Here we report a method to identify SARS-CoV-2 (COVID-19) virus RNA from purified RNA or cell lysis using loop-mediated isothermal amplification (LAMP) using a visual, colorimetric detection. This test was additionally verified using RNA samples purified from respiratory swabs collected from COVID-19 patients in Wuhan, China with equivalent performance to a commercial RT-qPCR test while requiring only heating and visual inspection. This simple and sensitive method provides an opportunity to facilitate virus detection in the field without a requirement for complex diagnostic infrastructure.

Effect of sublingual immunotherapy on the balance of Th17/Treg for children with mite, allergic asthma / 临床儿科杂志

Objective To explore the effect of sublingual immunotherapy (S-LIT) on the balance of Th17/Treg in children with mite allergic asthma. Methods All 124 cases with mite allergic asthma were randomly divided into two groups, the control group (62 cases) was treated with ladder type inhalation, and the treatment group (62 cases) was administered of dust mite drops on the basis of conventional treatment. The peripheral frequency of Th17 cell and Treg cell of all the subjects (before and after treatment) were detected by flow cytometry, the serum cytokine IL-10、IL-17 levels wer analyzed by ELISA. Results After 1 year treatment, the Th17 cells in treatment group (1.50 %±0.87 %) was significantly lower than that before treatment (3.39 % ± 1.58 %), and less than that of the control group after 1 year of treatment (2.42 %±1.32 %) (P <0.01);after 1 year treatment, the proportion of Treg cells in treatment group (4.05 %±1.36 %) was significantly higher than that before treatment (2.33 ± 0.81%), and more than that of the control group after 1 year of treatment (2.87 %± 0.87 %) (P <0.01). After 1 year treatment, the serum IL-10 levels in treatment group (64.76±27.79 pg/mL) was significantly higher than that before treatment (36.32 ± 11.53 pg/mL), and more than that of the control group after 1 years of treatment (50.32 ± 10.97 pg/mL) (P <0.01);after 1 year treatment, the serum IL-17 levels in treatment group (20.45±8.35 pg/mL) was significantly lower than that before treatment (86.48 ± 28.19 pg/mL), and significantly less than that of the control group after 1 year of treatment (46.32 ± 12.43 pg/mL) (P <0.01). After 1 year treatment, the Childhood Asthma Control Test (C-ACT)score in the treatment group (24.35 ± 8.47) was significantly higher than the control group (20.13 ± 6.86) (P <0.05). Conclusions SLIT can decrease airway inflammation and correct the imbalance of Th17/Treg in children with mite, allergic asthma.

Analysis on clinical characteristics of primary hypothyroidism-induced mental disorder / 中南大学学报(医学版)

OBJECTIVE@#To summarize the clinical features and early diagnostic evidence for mental disorders due to primary hypothyroidism after primary overactive thyroid surgery or 131I therapy. 
@*METHODS@#The retrospective analysis was conducted on 15 patients in terms of the clinical features with primary hypothyroidism-induced mental disorder after primary overactive thyroid surgery or 131I therapy. The data regarding past history of hyperthyroidism parallel operation or 131I treatment, thyroid function biochemical indexes were collected.
@*RESULTS@#The free triiodothyronine (FT3) and free thyroxine (FT4) were decreased, while thyroid-stimulating hormone (TSH) was increased. The clinical symptoms included lazy, fatigue, and mood swings accompanied by mental retardation, behavioral disorders, hallucinations and delusions. Particularly, the severe patients were of disturbance of consciousness. 
@*CONCLUSION@#The clinical features of primary hypothyroidism-induced mental disorder are diverse and variable. It is not difficult to diagnose the mental disorder if the attention is paid on the medical history inquiry and thyroid function tests. However, it is easy to be misdiagnosed and missed diagnosis.

Rapid Detection of Filoviruses by Real-time TaqMan Polymerase Chain Reaction Assays / 中国病毒学·英文版

Ebola virus (EBOV) and Marburg virus (MARV) are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics.To date,there is no specific laboratory diagnostic test in China,while there is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures.In this study,the TaqMan RT-PCR assays targeting the nucleoprotein genes of the Zaire Ebolavirus (ZEBOV) and MARV were developed and their sensitivities and specificities were investigated.Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109,corresponding to the threshold of a standard RNA transcript.The results showed that there were about 1010 RNA copies per milliliter of virus culture supernatant,equivalent to 10,000 RNA molecules per infectious virion,suggesting the presence of many non-infectious particles.These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable for future surveillance and specific diagnosis of ZEBOV and MARV in China.

Laboratory Detection and Diagnosis of Filoviruses / 中国病毒学·英文版

Ebola virus(EBOV)and Marburg virus(MARV),belonging to the Filoviridae family,emerged four decades ago and caused severe viral hemorrhagic fever in human and other primates.As high as 50-90% mortality,filoviruses can cause significant threats to public health.However,so far no specific and efficient vaccine has been available,nor have other treatment methods proved to be effective.It is of great importance to detect these pathogens specific,rapidly and sensitively in order to control future filovirus outbreaks.Here,recent progresses in the development of detection and diagnosis methods for EBOV and MARV are summarized.

Significance of serum cystatin-C, β2-mierogiobulin and renal hemodynamic in diagnosis of Henoch-Schoenlein purpura nephritis / 中国医师进修杂志

Objective To explore the changes and clinical significance of serum cystatin-C, β2- mieroglobulin (β2-MG) and renal hemodynamic in children with Henoch-Schoenlein purpura(HSP). Method Forty-six patients with HSP are in HSP group, 40 healthy children are in control group. Serum cystatin-C was determined by enzyme-linked immunosorbent assay, β2-MG was detected by radioimmunoassay, renal hemodynamie was detected by colour Doppler ultrasound. Results Serum cystafin-C and β2-MG in HSP group [(3.96±1.52 ), (2.74±0.82)mg/L] were higher than those in control group [(1.67±0.61), (1.89±0.47)mg/L] (P<0.01). Frequenee spectra showed high velocity and resistance, and maximum crest flow rate[(1.068±0.348) m/s] and resistance index (0.894±0.125) in systolic phase of main renal arteries were obviously higher in HSP group than those in control group [(0.859±0.357) m/s and 0.726±0.078] (P<0.05). Conclusions The level of serum cystatin-C and the change of renal hemedynamie can act as the significant indicators of early diagnosis of HSP nephritis.

Production and characterization of monoclonal antibodies to nucleoprotein of Marburg virus.

Monoclonal antibodies (MAbs) against the nucleoprotein of Marburg virus (MARV-NP) with high specificity and activity would be useful for the diagnosis of MARV. In this report, a recombinant MARV-NP was successfully expressed by an Escherichia coli expression system. After immunization and cell fusion, three mouse hybridomas (1H4, 2G1, and 3B5) producing MAbs to MARV-NP were established. The MAbs obtained were fully characterized using indirect ELISA and Western blot analysis. The ELISA results showed that the MAbs' titers were in the range of 1:6.400 x 10(3) - 1:1.280 x 10(4) in culture fluids, and 1:1.024 x 10(6) - 1:8.192 x 106 in ascitic fluids. The isotypes of the three MAbs were tested to be IgG1(kappa) and all the MAbs recognized the same antigenic epitope. Western blot analyses demonstrated that all the MAbs were directed against MARV-NP with the affinity constants (Kaff) of about 1.100 x 10(9) M(-1), 1.235 x 10(9) M(-1), and 1.408 x 10(9) M(-1) for the MAbs 1H4, 2G1, and 3B5, respectively.