Slime molds (Myxomycetes) causing a "disease" in crop plants and cultivated mushrooms.

Myxomycetes (plasmodial slime molds) are eukaryotic protist predators that are associated with wood, leaf litter, and soil in forests, where they feed on bacteria, protozoans, and (to a more limited extent) fungi. The health of crop plants is essential because they represent a primary food source for humans. However, when myxomycetes produce numerous fruiting bodies on the stems and leaves of crop plants, which is herein referred to as a myxomycete colonization, this has the potential of interfering with plant photosynthesis, transpiration and respiration by blocking out light and covering stomata. Myxomycetes are not pathogens, but their occurrence on plants can be mistakenly interpreted as some type of infection. However, this phenomenon has been largely ignored. This paper provides a comprehensive overview of the taxonomic and economic diversity of the organisms involved in myxomycete colonization. In addition, the various types of myxomycete colonization reported in the literature are described and discussed, a number of images provided, and cultural and chemical prevention and control measures are summarized. The latter should be of significant relevance for local production of crops and plant protective stations. While myxomycetes are not pathogens of crop plants, some species can seriously impact commercially grown mushrooms. Reports of myxomycetes affecting mushrooms are also described in this paper.

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. CONCLUSION: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

Preamplification-free ultra-fast and ultra-sensitive point-of-care testing via LwaCas13a.

CRISPR based nucleic acid detection technology provides a deployable approach to point of care testing. While, there remain challenges limiting its practical applications, such as the need for pre-amplification and the long turnaround time. Here, we present a self-cascade signal amplification method with LwaCas13a and an artificially designed "U" rich RNA of stem-loop structure (URH) for pre-amplification-free ultra-fast and ultra-sensitive point-of-care testing (PASSPORT). The PASSPORT system contains: URH, crRNA targeted the URH, crRNA targeted the interesting RNA, fluorescent RNA reporter and LwaCas13a. The assay realized the detection of 100 copies/mL, within 5 min. The PASSPORT platform was further adopted for the detection of marker gene from SASR-CoV-2 and Severe fever with thrombocytopenia syndrome virus (SFTSV), respectively, and 100% accuracy for the analysis of clinical specimens (100 SASR-CoV-2 specimens and 16 SFTSV specimens) was obtained. Integrated with a lateral flow assay device, this assay could provide an alternative platform for the development of point of care testing (POCT) biosensors. PASSPORT has the potential to enable sensitive, specific, user-friendly, rapid, affordable, equipment-free and point-of-care testing for the purpose of large-scale screening and in case of epidemic outbreak.

Fast and practical method for underwater stereo vision calibration based on ray-tracing.

The perspective camera model has difficulty handling refracted light in the underwater environment. To achieve accurate and convenient calibration in large underwater scenes, we propose a method based on the underwater refractive camera model in this paper. First, the initial values of the refraction parameters are solved using refraction coplanarity constraints. Then the initial values are optimized nonlinearly using co-point constraints, which simplifies the optimization process of existing methods. In the field of view of 200m m×200m m, the experiment results show that the reconstruction accuracy of the proposed method can reach below 0.02 mm, and it is equally effective in the case of sparse calibration.

TLTC, a T5 exonuclease-mediated low-temperature DNA cloning method.

Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease-mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp-25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into Escherichia coli to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments.

Applications of Programmable Endonucleases in Sequence- and Ligation-Independent Seamless DNA Assembly.

Programmable endonucleases, such as Cas (Clustered Regularly-Interspaced Short Repeats-associated proteins) and prokaryotic Argonaute (pAgo), depend on base pairing of the target DNA with the guide RNA or DNA to cleave DNA strands. Therefore, they are capable of recognizing and cleaving DNA sequences at virtually any arbitrary site. The present review focuses on the commonly used in vivo and in vitro recombination-based gene cloning methods and the application of programmable endonucleases in these sequence- and ligation-independent DNA assembly methods. The advantages and shortcomings of the programmable endonucleases utilized as tools for gene cloning are also discussed in this review.

Severe hyperlipidemia pancreatitis induced by taking tamoxifen after breast cancer surgery-Case report.

Introduction: The research investigates the mechanism, diagnosis, treatment, and subsequent endocrine therapy of severe pancreatitis induced by tamoxifen treatment in patients who have undergone breast cancer surgery. Case presentation: We studied two cases of breast cancer in whom severe acute pancreatitis developed after taking tamoxifen for endocrine therapy in our hospital. A brief literature review was provided to analyze the causes, clinical manifestations, treatment process, and prognosis of severe acute pancreatitis. Both cases involved patients with severe hyperlipidemic pancreatitis. After conservative treatment, none of them died. Pancreatitis did not recur after changing endocrine therapy drugs. Discussion/conclusion: Endocrine therapy with tamoxifen in breast cancer patients can induce hyperlipidemia, which can subsequently cause severe pancreatitis. The treatment of severe pancreatitis should strengthen the regulation of blood lipids. The application of low-molecular-weight heparin combined with insulin therapy can rapidly lower blood lipids. Involved treatments, including acid suppression, enzyme suppression, and peritoneal dialysis, can accelerate the recovery of pancreatitis and reduce the occurrence of serious complications. Patients with severe pancreatitis should not continue to use tamoxifen for endocrine therapy. To complete follow-up endocrine therapy, switching to a steroidal aromatase inhibitor is better if the situation allows it.

Somatic nuclear blebbing in Caenorhabditis elegans is not a feature of organismal aging but a potential indicator of germline proliferation in early adulthood.

Abnormal nuclear morphology is suggested to be a hallmark of aging and one such abnormality is nuclear blebbing. However, little is known about whether and how nuclear blebbing participates in animal aging, and what regulates it. In this study, we show that the frequency of nuclear blebbing in the hypodermis increases during aging in wild-type C. elegans. These nuclear blebs are enveloped by the nuclear lamina, the inner and the outer nuclear membrane, and 42% of them contain chromatin. Although nuclear blebbing could lead to DNA loss if chromatin-containing blebs detach and fuse with lysosomes, we find by time-lapse imaging that nuclear blebs rarely detach, and the estimated lifetime of a nuclear bleb is 772 h or 32 days. The amount of DNA lost through nuclear blebbing is estimated to be about 0.1% of the total DNA loss by adult Day 11. Furthermore, the frequency of nuclear blebbing does not correlate with the rate of aging in C. elegans. Old age does not necessarily induce nuclear blebbing, neither does starvation, heat stress, or oxidative stress. Intriguingly, we find that proliferation of germ cells promotes nuclear blebbing.

Improving the activity and thermostability of PETase from Ideonella sakaiensis through modulating its post-translational glycan modification.

The large-scale preparation of Polyehylene terephthalate (PET) hydrolysing enzymes in low-cost is critical for the biodegradation of PET in industry. In the present study, we demonstrate that the post-translational glycosylation of Pichia pastoris makes it a remarkable host for the heterologous expression of PETase from Ideonella sakaiensis 201-F6 (IsPETase). Taking advantage of the abundant N- and O-linked glycosylation sites in IsPETase and the efficient post-translational modification in endoplasmic reticulum, IsPETase is heavily glycosylated during secretory expression with P. pastoris, which improves the specific activity and thermostability of the enzyme dramatically. Moreover, the specific activity of IsPETase increased further after the bulky N-linked polysaccharide chains were eliminated by Endo-ß-N-acetylglucosaminidase H (Endo H). Importantly, the partially deglycosylated IsPETase still maintained high thermostability because of the remaining mono- and oligo-saccharide residues on the protein molecules. Consequently, the partially deglycosylated IsPETase was able to be applied at 50 °C and depolymerized raw, untreated PET flakes completely in 2 to 3 days. This platform was also applied for the preparation of a famous variant of IsPETase, Fast-PETase, and the same result was achieved. Partially deglycosylated Fast-PETase demonstrates elevated efficiency in degrading postconsumer-PET trays under 55 °C than 50 °C, the reported optimal temperature of Fast-PETase. The present study provides a strategy to modulate thermostable IsPETase through glycosylation engineering and paves the way for promoting PET biodegradation from laboratories to factories.

Fusion and expansion of vitellogenin vesicles during Caenorhabditis elegans intestinal senescence.

Some of the most conspicuous aging phenotypes of C. elegans are related to post-reproductive production of vitellogenins (Vtg), which form yolk protein (YP) complexes after processing and lipid loading. Vtg/YP levels show huge increases with age, and inhibition of this extends lifespan, but how subcellular and organism-wide distribution of these proteins changes with age has not been systematically explored. Here, this has been done to understand how vitellogenesis promotes aging. The age-associated changes of intestinal vitellogenin vesicles (VVs), pseudocoelomic yolk patches (PYPs), and gonadal yolk organelles (YOs) have been characterized by immuno-electron microscopy. We find that from reproductive adult day 2 (AD 2) to post-reproductive AD 6 and AD 9, intestinal VVs expand from 0.2 to 3-4 µm in diameter or by >3000 times in volume, PYPs increase by >3 times in YP concentration and volume, while YOs in oocytes shrink slightly from 0.5 to 0.4 µm in diameter or by 49% in volume. In AD 6 and AD 9 worms, mislocalized YOs found in the hypodermis, uterine cells, and the somatic gonadal sheath can reach a size of 10 µm across in the former two tissues. This remarkable size increase of VVs and that of mislocalized YOs in post-reproductive worms are accompanied by extensive fusion between these Vtg/YP-containing vesicular structures in somatic cells. In contrast, no fusion is seen between YOs in oocytes. We propose that in addition to the continued production of Vtg, excessive fusion between VVs and mislocalized YOs in the soma worsen the aging pathologies seen in C. elegans.

Circulating Tumor-Derived Endothelial Cells: An Effective Biomarker for Breast Cancer Screening and Prognosis Prediction.

Background: Circulating tumor-derived endothelial cell (CTEC) is a new potential tumor biomarker to be associated with cancer development and treatment efficacy. However, few evidences are available for breast cancer. Methods: Eighty-nine breast cancer patients were recruited, and preoperative and postoperative blood samples were collected. Besides, 20 noncancer persons were enrolled as controls. An improved subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) method was adopted to codetect CD31+ aneuploid CTEC and CD31- aneuploid circulating tumor cell (CTC). Then, the clinical significance of CTCs and CTECs on breast cancer screening and prognosis prediction was evaluated and compared. Results: The positive rate of CTCs and CTECs in newly diagnosed breast cancer patients was 68.75% and 71.88%. Among detected aneuploid circulating rare cells, CTEC accounts for a greater proportion than CTC in breast cancer patients. CTEC-positive rate and level were significantly higher in breast cancer patients with lymph node metastasis (LNM) than those without LNM (P=0.043), while there was no significant difference in CTC. CTEC (area under the curve, AUC = 0.859) had better performance than CTC (AUC = 0.795) to distinguish breast cancer patients from controls by receiver operator characteristic curve analysis. Preoperative CTEC count ≥ 2 was a significant risk factor for reducing PFS of breast cancer patients. Conclusions: CTECs may function as a reliable supplementary biomarker in breast cancer screening and prognosis prediction.

Qualitative and Quantitative Real-Time PCR Methods for Assessing False-Positive Rates in Genetically Modified Organisms Based on the Microbial-Infection-Linked HPT Gene.

The hygromycin phosphotransferase (HPT) gene as a selective marker is normally used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in seeds, food, and feed materials. Nevertheless, if researchers only focus on the HPT gene, it is difficult to distinguish genetically modified (GM) crops from microbial infection, leading to miscalculation of the rate of GM materials in a given sample set. Here, we cloned the 7259 bp sequence carrying the HPT gene from soybean sprouts using the genome walking strategy. BLAST analysis revealed that this sequence was derived from plasmids naturally occurring in microorganisms, such as Escherichia coli, Klebsiella pneumoniae or Salmonella sp. Using the reconstructed plasmid pFP-hpt, qualitative PCR and quantitative real-time PCR (qPCR) methods were established, and 261 bp and 156 bp products were produced. The specificity of these assays was assessed against related pFP-hpt plasmids, plant species with important agronomic traits, and GM crops containing the HPT gene. No unexpected results were observed between samples using these qualitative PCR and qPCR methods. The sensitivity of this qualitative PCR assay was determined at 20 copies, while the limit of detection (LOD) and limit of quantification (LOQ) of qPCR were both 5 copies per reaction. Our in-house validation indicated that the amplification efficiency, linearity, and repeatability of this qPCR assay were in line with performance requirements. Furthermore, a qualitative and quantitative duplex PCR showed high reliability for the simultaneous detection of the HPT gene in a plant sample and environmental micro-organisms harboring the HPT gene in one PCR reaction. These qualitative PCR and qPCR assays were able to differentiate between plants infected with E. coli harboring the HPT gene from GM plants, indicating that these two methods are broadly applicable for routine GMO testing.

Flow characteristics and formation optimization of vortex ring air supply.

The vortex rings ventilation (VRV) is a new type of air supply system comprising vortex rings. Compared with an air supply jet, a vortex ring reduces the loss of fresh air during transportation because of its stable structure. However, during the formation of the vortex ring, it entrains the ambient air and reduces the fresh air in the vortex ring. In this study, a vortex ring generator with a fresh air cavity is proposed to form confined vortex rings. This improved the fresh air ratio of the VRV. Based on previous experiments, a piston-orifice axisymmetric model with a dynamic grid was developed to form an air vortex ring. The flow characteristics of free and confined vortex rings during the generation stage were studied. First, the evolution of free vortex rings with different stroke lengths was studied, and the optimal piston stroke length and radial constraint size were determined. Subsequently, the mixing ratios of the free and confined vortex rings were compared and analyzed. The results showed that the mixing ratio of the confined vortex ring formed by the fresh air cavity in the formation stage was zero. Moving to the location of 4.9 times the orifice diameter ( D 0 $$ {D}_0 $$ ), reduced the mixing ratio of the confined vortex ring by 77.78% compared with that of the free vortex ring. In addition, the influence of three inner diameters and four outlet diameters of fresh air cavities on the vortex rings was studied to optimize the size of the vortex ring generator. The results showed that the inner diameter of the fresh air cavity was greater than 3 D 0 $$ 3{D}_0 $$ and that an outlet diameter greater than 2.5 D 0 $$ 2.5{D}_0 $$ had little influence on the vortex rings.

Knockdown of CD146 promotes endothelial-to-mesenchymal transition via Wnt/ß-catenin pathway.

PURPOSE: Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) proteins and leads to the maladaptive changes in myocardium. Endothelial cells (ECs) undergoing mesenchymal transition contributes to the occurrence and development of cardiac fibrosis. CD146 is an adhesion molecule highly expressed in ECs. The present study was performed to explore the role of CD146 in modulating endothelial to mesenchymal transition (EndMT). METHODS: C57BL/6 mice were subjected to subcutaneous implantation of osmotic minipump infused with angiotensin II (Ang Ⅱ). Adenovirus carrying CD146 short hairpin RNA (shRNA) or CD146 encoding sequence were infected into cultured human umbilical vein endothelial cells (HUVECs) followed by stimulation with Ang II or transforming growth factor-ß1 (TGF-ß1). Differentially expressed genes were revealed by RNA-sequencing (RNA-Seq) analysis. Gene expression was measured by quantitative real-time PCR, and protein expression and distribution were determined by Western blot and immunofluorescence staining, respectively. RESULTS: CD146 was predominantly expressed by ECs in normal mouse hearts. CD146 was upregulated in ECs but not fibroblasts and myocytes in hearts of Ang II-infused mice and in HUVECs stimulated with Ang Ⅱ. RNA-Seq analysis revealed the differentially expressed genes related to EndMT and Wnt/ß-catenin signaling pathway. CD146 knockdown and overexpression facilitated and attenuated, respectively, EndMT induced by Ang II or TGF-ß1. CD146 knockdown upregulated Wnt pathway-related genes including Wnt4, LEF1, HNF4A, FOXA1, SOX6, and CCND3, and increased the protein level and nuclear translocation of ß-catenin. CONCLUSIONS: Knockdown of CD146 exerts promotional effects on EndMT via activating Wnt/ß-catenin pathway and the upregulation of CD146 might play a protective role against EndMT and cardiac fibrosis.

Site-Directed Mutagenesis Method Mediated by Cas9.

This study presents an in vitro CRISPR/Cas9-mediated mutagenic (ICM) system that allows rapid construction of designed mutants or site-saturation mutagenesis libraries in a PCR-independent manner. The plasmid DNA is double digested with Cas9 bearing specific single guide RNAs to remove the target nucleotides. Next, T5 exonuclease excises both 5'-ends of the linearized plasmid to generate homologous regions of approximately 15 nt. Subsequently, a short dsDNA of approximately 30-50 bp containing the desired mutation cyclizes the plasmid through base pairing and introduces the mutation into the plasmid. The gaps are repaired in Escherichia coli host cells after transformation. This method is highly efficient and accurate. Both single and multiple site-directed mutagenesis can be successfully performed, especially to large sized plasmids. This method demonstrates the great potential for creating high-quality mutant libraries in directed evolution as an alternative to PCR-based saturation mutagenesis, thus facilitating research on synthetic biology.

Dental Implant Navigation System Based on Trinocular Stereo Vision.

Traditional dental implant navigation systems (DINS) based on binocular stereo vision (BSV) have limitations, for example, weak anti-occlusion abilities, as well as problems with feature point mismatching. These shortcomings limit the operators' operation scope, and the instruments may even cause damage to the adjacent important blood vessels, nerves, and other anatomical structures. Trinocular stereo vision (TSV) is introduced to DINS to improve the accuracy and safety of dental implants in this study. High positioning accuracy is provided by adding cameras. When one of the cameras is blocked, spatial positioning can still be achieved, and doctors can adjust to system tips; thus, the continuity and safety of the surgery is significantly improved. Some key technologies of DINS have also been updated. A bipolar line constraint algorithm based on TSV is proposed to eliminate the feature point mismatching problem. A reference template with active optical markers attached to the jaw measures head movement. A T-type template with active optical markers is used to obtain the position and direction of surgery instruments. The calibration algorithms of endpoint, axis, and drill are proposed for 3D display of the surgical instrument in real time. With the preoperative path planning of implant navigation software, implant surgery can be carried out. Phantom experiments are carried out based on the system to assess the feasibility and accuracy. The results show that the mean entry deviation, exit deviation, and angle deviation are 0.55 mm, 0.88 mm, and 2.23 degrees, respectively.

Degradation of CP4-EPSPS with a Psychrophilic Bacterium Stenotrophomonas maltophilia 780.

CP4-EPSPS (Agrobacterium sp. strain CP4 5-enolpyruvylshikimate-3-phosphate synthase) protein showed remarkable thermostability and was highly resistant to proteases, such as trypsin. In order to eliminate the pollution of CP4-EPSPS from the accumulated straws to the surrounding environment during the winter, the present study investigated the extracellular proteases of 21 psychrophilic strains isolated from the south polar region. The results indicated that Stenotrophomonas maltophilia 780 was able to degrade CP4-EPSPS at 18 °C efficiently. Further study indicated that it was able to grow in the extract of Roundup Ready soybean at 18 °C, with CP4-EPSPS degraded to an undetectable level within 72 h. The extracellular proteases of Stenotrophomonas maltophilia 780 are thermo-sensitive, with an optimal temperature of 65 °C. The genomic sequencing result indicated that this strain had more than a hundred putative protease and peptidase coding genes, which may explain its high capability in decomposing CP4-EPSPS.

Low voltage optical fiber positioner robot based on minimum inductance hollow cup motors.

With the further transformation of The Large Sky Area Multi-Object Fiber Spectroscopic Telescope, the new generation of fiber positioner robot chooses a 4 mm hollow cup motor with minimum phase inductance. Because the load of the fiber positioner robot is constant and the inertia of the motor is very small, an open loop positioning control method based on Space Vector Pulse Width Modulation is proposed, and the specific open loop parameters are directly tuned by relevant experimental strategies. The critical factors of the open loop driving mode are discussed in detail from four aspects: subdivision, fundamental frequency, wave generation mode and peak current. Based on the actual fiber positioner robot, the hardware driver and assessment platform are built. The positioning tests show that the method proposed is practical and effective, and meets the precision positioning demand of the new generation optical fiber positioner robot.

High Precision Optical Tracking System Based on near Infrared Trinocular Stereo Vision.

A high precision optical tracking system (OTS) based on near infrared (NIR) trinocular stereo vision (TSV) is presented in this paper. Compared with the traditional OTS on the basis of binocular stereo vision (BSV), hardware and software are improved. In the hardware aspect, a NIR TSV platform is built, and a new active tool is designed. Imaging markers of the tool are uniform and complete with large measurement angle (>60°). In the software aspect, the deployment of extra camera brings high computational complexity. To reduce the computational burden, a fast nearest neighbor feature point extraction algorithm (FNNF) is proposed. The proposed method increases the speed of feature points extraction by hundreds of times over the traditional pixel-by-pixel searching method. The modified NIR multi-camera calibration method and 3D reconstruction algorithm further improve the tracking accuracy. Experimental results show that the calibration accuracy of the NIR camera can reach 0.02%, positioning accuracy of markers can reach 0.0240 mm, and dynamic tracking accuracy can reach 0.0938 mm. OTS can be adopted in high-precision dynamic tracking.

PfAgo-based detection of SARS-CoV-2.

In the present study, we upgraded Pyrococcus furiosus Argonaute (PfAgo) mediated nucleic acid detection method and established a highly sensitive and accurate molecular diagnosis platform for the large-scale screening of COVID-19 infection. Briefly, RT-PCR was performed with the viral RNA extracted from nasopharyngeal or oropharyngeal swabs as template to amplify conserved regions in the viral genome. Next, PfAgo, guide DNAs and molecular beacons in appropriate buffer were added to the PCR products, followed by incubating at 95 °C for 20-30 min. Subsequently, the fluorescence signal was detected. This method was named as SARS-CoV-2 PAND. The whole procedure is accomplished in approximately an hour with the using time of the Real-time fluorescence quantitative PCR instrument shortened from >1 h to only 3-5 min per batch in comparison with RT-qPCR, hence the shortage of the expensive Real-time PCR instrument is alleviated. Moreover, this platform was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The diagnostic results of clinic samples with SARS-CoV-2 PAND displayed 100% consistence with RT-qPCR test.