Medical diagnostic value of digital PCR (dPCR): A systematic review

Digital polymerase chain reaction (dPCR) is an emerging technique for the absolute quantification of target nucleic acids. dPCR got attention as a precise quantification tool in preclinical research, particularly when used to detect genetic mutations and result in highly precise measurements. In dPCR, the statistic of Poisson distribution was followed for the random distribution of molecules in different partitions, which is essential for dPCR quantification. Amplified target sequences in different partitions are identified by fluorescence and each partition functions as a separate PCR microreactor. Without the need for calibration, the percentage of PCR-positive partitions is sufficient to estimate the concentration of the target sequence. The present revolution in digital quantification was made possible by advancements in microfluidics, which provided effective partitioning techniques. In this paper, the contrast of the underlying ideas of quantitative real-time PCR with dPCR for the measurement of nucleic acids quantity Polymerase chain reaction (q-PCR). This review study briefly introduced the background of dPCR and compared different types of PCR, particularly the quantity of real-time qPCR and digital PCR. The fundamental concept of dPCR is also explained and also briefly compares the advantages of dPCR over qPCR and analyzes the applications of dPCR as a diagnostic tool for cancer and different types of viral species.

Ultrafast and absolute quantification of SARS-CoV-2 on food using hydrogel RT-LAMP without pre-lysis.

With rapid growing of environmental contact infection, more and more attentions are focused on the precise and absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus on cold chain foods via point-of-care test (POCT). In this work, we propose a hydrogel-mediated reverse transcription loop-mediated isothermal amplification (RT-LAMP) for ultrafast and absolute quantification of SARS-CoV-2. Cross-linked hydrogel offers opportunities for digital single molecule amplification in nanoconfined spaces, facilitating the virus lysis, RNA reverse transcription and amplification process, which is about 3.4-fold faster than conventional bulk RT-LAMP. Ultrafast quantification of SARS-CoV-2 is accomplished in 15 min without virus pre-lysis and RNA extraction. The sensitivity can accurately quantify SARS-CoV-2 down to 0.5 copy/µL. Furthermore, the integrated system has an excellent specificity, reproducibility and storage stability, which can be also used to test SARS-CoV-2 on various cold chain fruits. The developed ultrafast and simple hydrogel RT-LAMP will be an enormous potential for surveillance of virus or other hazardous microbes in environmental, agricultural and food industry.

An integrated digital PCR system with high universality and low cost for nucleic acid detection.

Digital PCR is the most advanced PCR technology. However, due to the high price of the digital PCR analysis instrument, this powerful nucleic acid detection technology is still difficult to be popularized in the general biochemistry laboratory. Moreover, one of the biggest disadvantages of commercial digital PCR systems is the poor versatility of reagents: each instrument can only be used for a few customized kits. Herein, we built a low-cost digital PCR system. The system only relies on low-cost traditional flat-panel PCR equipment to provide temperature conditions for commercial dPCR chips, and the self-made fluorescence detection system is designed and optically optimized to meet a wide range of reagent requirements. More importantly, our system not only has a low cost (<8000 US dollars) but also has a much higher universality for nucleic acid detection reagents than the traditional commercial digital PCR system. In this study, several samples were tested. The genes used in the experiment were plasmids containing UPE-1a fragment, TP53 reference DNA, hepatitis B virus DNA, leukemia sample, SARS-COV-2 DNA, and SARS-COV-2 RNA. Under the condition that DNA can be amplified normally, the function of the dPCR system can be realized with simpler and low-price equipment. Some DNA cannot be detected by using the commercial dPCR system because of the special formula when it is configured as the reaction solution, but these DNA fluorescence signals can be clearly detected by our system, and the concentration can be calculated. Our system is more applicable than the commercial dPCR system to form a new dPCR system that is smaller and more widely applicable than commercially available machinery.

A fast and sensitive absolute quantification assay for the detection of SARS-CoV-2 peptides using parallel reaction monitoring mass spectrometry.

The on-going SARS-CoV-2 (COVID-19) pandemic has called for an urgent need for rapid and high-throughput methods for mass testing and early detection, prevention as well as surveillance of the disease. We investigated whether targeted parallel reaction monitoring (PRM) quantification using high resolution Orbitrap instruments can provide the sensitivity and speed required for a high-throughput method that could be used for clinical diagnosis. We developed a high-throughput and sensitive PRM-MS assay that enables absolute quantification of SARS-CoV-2 nucleocapsid peptides with short turn-around times by using isotopically labelled synthetic SARS-CoV-2 concatenated peptides. We established a fast and high-throughput S-trap-based sample preparation method and utilized it for testing 25 positive and 25 negative heat-inactivated clinical nasopharyngeal swab samples for SARS-CoV-2 detection. The method was able to differentiate between negative and some of the positive patients with high viral load. Moreover, based on the absolute quantification calculations, our data show that patients with Ct values as low as 17.8 correspond to NCAP protein amounts of around 7.5 pmol in swab samples. The present high-throughput method could potentially be utilized in specialized clinics as an alternative tool for detection of SARS-CoV-2 but will require enrichment of viral proteins in order to compete with RT-qPCR.

Multiplex Digital Polymerase Chain Reaction Technology and Its Application

Digital polymerase chain reaction (PCR), as a nucleic acid detection technology with wide application prospect, has become one of the most accurate nucleic acid detection technology at present. Multiplex detection is an important direction for the development of digital PCR technique. With the development of microfluidic technology, multiplex digital PCR technique has become more and more mature. This paper reviewed the research progresses of multiplex digital PCR in recent years, especially summarized the implementation of multiplex digital PCR technique in the past five years, and introduced the application of multiplex digital PCR technique in hot areas such as liquid biopsy, transgenic detection, and SARS-Cov-2 detection. Finally, the issues and challenges faced by multiplex digital PCR technique were discussed and the future direction of the technology was foreseen.

Investigation of SARS-CoV-2 on Ocular Surface of Coronavirus Disease 2019 Patients Using One-Step Reverse-Transcription Droplet Digital PCR.

PURPOSE: This study detects SARS-CoV-2 in the ocular surface through one-step reverse-transcription droplet digital PCR (one-step RT-ddPCR) and evaluates the possibility of the ocular surface as a possible transmission route. METHODS: A single-center prospective observational study was designed to investigate the viral loads in ocular surface. Specimens including the conjunctival swabs, nasopharyngeal swabs and blood were synchronously collected at a single time point for all COVID-19 patients. SARS-CoV-2 loads in nasopharyngeal swabs were tested by real-time polymerase chain reaction (PCR); the blood samples and conjunctival swabs were tested by real-time PCR and one-step RT-ddPCR. RESULTS: Sixty-eight COVID-19 patients confirmed by nasopharyngeal real-time PCR were recruited. In the single time point test, 40 cases showed positive SARS-CoV-2 detection in either the blood, tears, or nasopharynx, of which four cases were triple-positive, 10 were dual-positive, and 26 were single-positive. The positive rate of nasopharyngeal swab real-time PCR test was 22.1% (15/68). The positive rate of blood and conjunctival swabs by one-step RT-ddPCR was 38.2% (26/68) and 25% (17/68), respectively, whereas real-time PCR was all negative. Positive conjunctival swabs were significantly correlated with positive nasopharyngeal swabs (P = 0.028). The sampling lags from illness onset to sampling day in 3 out of 4 triple-positive patients and in 9 out of 10 dual-positive patients were respectively less than 9 days and less than 20 days. CONCLUSION: Our results indicate that the positive rate of SARS-CoV-2 on the ocular surface is much higher than expected. Transmission possibility through the ocular surface may be greatly underestimated.

Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids.

Rapid diagnostics of adventitious agents in biopharmaceutical/cell manufacturing release testing and the fight against viral infection have become critical. Quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in 1 h but suffer from inter-site variability. Absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application. Here, we report a RApid DIgital Crispr Approach (RADICA) for absolute quantification of nucleic acids in 40-60 min. Using SARS-CoV-2 as a proof-of-concept target, RADICA allows for absolute quantification with a linear dynamic range of 0.6-2027 copies/µL (R2 value > 0.99), high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA's versatility is validated against other targets such as Epstein-Barr virus (EBV) from human B cells and patients' serum. RADICA can accurately detect and absolutely quantify EBV DNA with similar dynamic range of 0.5-2100 copies/µL (R2 value > 0.98) in 1 h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based detection. RADICA therefore enables rapid and sensitive absolute quantification of nucleic acids which can be widely applied across clinical, research, and biomanufacturing areas.

Quantitative Proteomic Analysis of Porcine Intestinal Epithelial Cells Infected with Porcine Deltacoronavirus Using iTRAQ-Coupled LC-MS/MS.

Porcine deltacoronavirus (PDCoV) is an emergent enteropathogenic coronavirus associated with swine diarrhea. Porcine small intestinal epithelial cells (IPEC) are the primary target cells of PDCoV infection in vivo. Here, isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantitatively identify differentially expressed proteins (DEPs) in PDCoV-infected IPEC-J2 cells. A total of 78 DEPs, including 23 upregulated and 55 downregulated proteins, were identified at 24 h postinfection. The data are available via ProteomeXchange with identifier PXD019975. To ensure reliability of the proteomics data, two randomly selected DEPs, the downregulated anaphase-promoting complex subunit 7 (ANAPC7) and upregulated interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), were verified by real-time PCR and Western blot, and the results of which indicate that the proteomics data were reliable and valid. Bioinformatics analyses, including GO, COG, KEGG, and STRING, further demonstrated that a majority of the DEPs are involved in numerous crucial biological processes and signaling pathways, such as immune system, digestive system, signal transduction, RIG-I-like receptor, mTOR, PI3K-AKT, autophagy, and cell cycle signaling pathways. Altogether, this is the first study on proteomes of PDCoV-infected host cells, which shall provide valuable clues for further investigation of PDCoV pathogenesis.

GSH-ZnS Nanoparticles Exhibit High-Efficiency and Broad-Spectrum Antiviral Activities via Multistep Inhibition Mechanisms.

Despite the good biocompatibility and antibacterial activity of zinc sulfide nanoparticles (ZnS NPs), whether they possess antiviral activity is still unclear. Here, GSH-modified ZnS NPs (GSH-ZnS NPs) were synthesized and their significant antiviral activity was demonstrated using the Arteriviridae family RNA virus, porcine reproductive and respiratory syndrome virus (PRRSV), as a model. Mechanistically, GSH-ZnS NPs were shown to reduce PRRSV-induced ROS production to prevent PRRSV multiplication, with no activating effect on the interferon (IFN) signal pathway, the first defense line against virus infection. Furthermore, isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic analysis of GSH-ZnS NP-treated cells revealed the involvement of numerous crucial proteins in virus proliferation, with vitronectin (VTN) being confirmed as an efficient PRRSV antagonist here. Furthermore, GSH-ZnS NPs were found to have potent antiviral effects on the Herpesviridae family DNA virus, pseudorabies virus (PRV), the Coronaviridae family positive-sense RNA virus, porcine epidemic diarrhea virus (PEDV), and the Rhabdoviridae family negative-stranded RNA virus, vesicular stomatitis virus (VSV), indicating their broad-spectrum antiviral activity against viruses from different families with various genome types. Overall, GSH-ZnS NP is a prospective candidate for the development of antiviral nanomaterials and may serve as a model for investigation of potential host restriction factors in combination with proteomics.