Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number.

The outbreak of coronavirus disease 2019 (COVID-19) has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the virus causing COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal swab samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed by three different methods: (i) incubation with the TRIzol LS reagent for 10 min at room temperature, (ii) heating in a water bath at 56°C for 30 min, and (iii) high-temperature treatment, including autoclaving at 121°C for 20 min, boiling at 100°C for 20 min, and heating at 80°C for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of the nucleocapsid protein (N) gene and 39.85% of open reading frame (ORF) 1ab. For samples treated at 56°C for 30 min, the copy number of the N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. The viral RNA copy number dropped by 50 to 66% after heating at 80°C for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicate that inactivation reduced the quantity of detectable viral RNA and may cause false-negative results, especially in weakly positive cases. Thus, use of the TRIzol reagent rather than heat inactivation is recommended for sample inactivation, as the TRIzol reagent had the least effect on the RNA copy number among the tested methods.

Design, Dynamics, and Optimization of a 3-DoF Nonlinear Micro-Gyroscope by Considering the Influence of the Coriolis Force.

In this paper, we use the nonlinear hardening stiffness of drive mode deal with the contradiction between gain and bandwidth of the linear micro-gyroscope, to improve the bandwidth and gain in sense direction. Firstly, in order to adjust the distance between two resonant peaks, we changed an incomplete two-degree-of-freedom(2-DoF) sense mode system of the micro-gyroscope into a complete 2-DoF system. Afterward, according to the given nonlinear coefficient of stiffness of drive mode, the structure size of driving micro-beams was designed to obtain a nonlinear micro-gyroscope with controllable stiffness. Finally, we investigated the effects of peaks spacing, damping, and driving nonlinearity on gain and bandwidth, and the nonlinear micro-gyroscope was optimized by orthogonal experiment method and response surface method. The results reveal that the peaks spacing has a great influence on the gain and bandwidth of both linear and nonlinear micro-gyroscopes. The larger the peaks spacing, the lower the gain, but higher gain can be achieved when the resonant frequency of the drive mode is close to the lower-order resonant frequency of the sense mode. Driving nonlinearity leads to the response peak of the Coriolis force to have a hardening characteristic, thus forming a wide platform in the sense direction. Hardening of the response peak of the Coriolis force allows the micro-gyroscope to obtain a higher gain while the bandwidth of the sense mode is also greatly improved. In addition, parameter optimization can make the gain and bandwidth of the micro-gyroscope optimal. When the peaks spacing is small and the nonlinear stiffness coefficient is about 1012.2, under the premise that the gain is basically constant, the bandwidth of the sense mode increases about 1.76 times compared with the linear gyroscope. Damping can suppress the influence of nonlinearity in a micro-gyroscope system. Within a certain range, the frequency response of the nonlinear micro-gyroscope tends to be a linear system with the increase in damping, resulting in narrower bandwidth and lower gain.

Preparation of a Bombyx mori acetylcholinesterase enzyme reagent through chaperone protein disulfide isomerase co-expression strategy in Pichia pastoris for detection of pesticides.

The cholinesterase-based spectrophotometric methods for detection of organophosphate pesticides (OPs) and carbamate pesticides (CPs) have been proposed as a good choice for their high efficiency, simplicity and low cost. The enzyme, as a core reagent, is of great importance for the developed method. In this study, a protein disulfide isomerase (PDI) co-expression strategy in Pichia pastoris was employed to enhance the yield of recombinant Bombyx mori acetylcholinesterase 2 (rBmAChE2). Subsequently, the prepared enzyme reagent was used to detect the pesticides in real samples. The results showed that the co-expression of rBmAChE2 with PDI increased the enzyme activity of the supernatant and the yield of purified rBmAChE2 up to 60 U/mL and 6 mg/L respectively, both almost 5-fold higher than those of original recombinant strain. In addition, 5 g/L gelatin reagent could help to preserve nearly 90% of the rBmAChE2 activity for 90 days in 4°C and the limits of detections (LODs) of the rBmAChE2-based assay for 20 kinds of OPs or CPs ranged from 0.010 to 2.725 mg/kg, which were lower than most of indexes present in current Chinese National Standard (GB/T 5009.199-2003) or the maximum residue limits (GB 2763-2019). Furthermore, the detection results of 23 vegetable samples were verified by the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, which indicated that the rBmAChE2-based assay in this work is suitable for pesticide residues rapid detection.

Clinical evaluation of the effectiveness of fusion-induced asymmetric transcription assay-based reverse transcription droplet digital PCR for ALK detection in formalin-fixed paraffin-embedded samples from lung cancer.

BACKGROUND: Accurate detection of anaplastic lymphoma kinase (ALK) rearrangement is the prerequisite for anti-ALK therapy for the patient with non-small cell lung cancer (NSCLC). Fusion-induced asymmetric transcription assay (FIATA)-based reverse transcription droplet digital PCR (RT-ddPCR) was developed and performed for ALK status survey in NSCLC samples. METHODS: A total of 269 cases of formalin-fixed paraffin-embedded (FFPE) specimens from NSCLC, in which ALK status was confirmed by both fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), were analyzed by FIATA-based RT-ddPCR. RESULTS: In the ALK-positive group, the 3' ALK transcript copies range was 336.6-107 955.4, and the R3 [(the ratio of the 3' ALK transcript copy numbers to the internal reference gene transcript copy numbers) × 100] was 17.23-672.77. In the ALK-negative group, the 3' ALK transcript copies range was 3.7-1370.6, and the R3 range was 0.10-15.57. The lowest R3 level in the ALK-positive group was significantly higher than the highest R3 level in the ALK-negative group. A positive correlation between the proportion of cancer cells in the tissue section and ALK RNA expression level (R3) was found (P < 0.05). There was no relationship between the percentage of FISH positive cells or FISH positive signal patterns and R3 level of the ALK gene. Compared with FISH and IHC, the clinical sensitivity and specificity of FIATA-based RT-ddPCR for ALK detection were 100%, respectively. CONCLUSIONS: An absolute quantitative FIATA-based RT-ddPCR was developed and validated for ALK fusion detection in NSCLC. This method can rapidly, accurately, and objectively classify ALK types and help with individual therapy.

Evaluation of the clinical performance of single-, dual-, and triple-target SARS-CoV-2 RT-qPCR methods.

BACKGROUND: The coronavirus disease 2019 (COVID-19) has become a pandemic. Reverse transcription quantitative PCR (RT-qPCR) has played a vital role in the diagnosis of COVID-19, but the rates of false negatives is not ideal in dealing with this highly infectious virus. It is thus necessary to systematically evaluate the clinical performance of the single-, dual-, triple-target detection kits to guide the clinical diagnosis of this disease. METHODS: A series of reference materials calibrated by droplet digital PCR (ddPCR) and 57 clinical samples were used to evaluate the clinical performance of six single-, dual-, triple-target SARS-CoV-2 nucleic acid detection kits based on RT-qPCR. RESULTS: The dual-target kits, kit B and kit C had the highest and the lowest detection sensitivity, which was 125 copies/mL and 4000 copies/mL, respectively. Among the 57 clinical samples from patients with COVID-19, 47 were tested positive by the kit B, while 35, 29, 28, 30, and 29 were found positive by the kits A, C, D, E, and F, respectively. The number of targets in a detection kit is not a key factor affecting sensitivity, while the amount of sample loading may influence the performance of a detection kit. CONCLUSIONS: This study provides a guide when choosing or developing a nucleic acid detection kit for the diagnosis of COVID-19. Also, the absolute-quantification feature and high-sensitivity performance of ddPCR, suggesting that it can be used to review clinically suspected samples.