Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Chinese Academy of Sciences

University of Chinese Academy of Sciences

Sino-Africa Joint Research Center

Droplet digital polymerase chain reaction (ddPCR) is a third generation of polymerase chain reaction (PCR) that enables the exact quantification of nucleic acid targets within a sample. The capability of ddPCR to accurately detect and quantify low abundant targets has led to its fast-growing applications in detection of different pathogens. This review summarizes the ddPCR technology and its applications in tuberculosis diagnosis. From current studies including a total of 9 publications on the applications of ddPCR in tuberculosis research, it is clear that ddPCR technology offers enormous advantages, such as unparalleled sensitivity, high precision, and absolute quantification without a standard curve, over common molecular diagnostic platforms like the real-time quantification PCR. The latest study also showed that rapid drug susceptibility test of Mycobacterium tuberculosis in sputa could be achieved within 4 days. However, the high cost is the main limitation for its wide applications, especially in developing countries. As we near the vision 2030 goal for sustainable development and ending the tuberculosis epidemic by 2030, ddPCR techniques may help achieve this objective and many more discussed in the UNGA-HLM-TB.

Droplet digital PCR applications in the tuberculosis world.

Mosquito-borne diseases, including arbovirus-related diseases, make up a large proportion of infectious disease cases worldwide, causing a serious global public health burden with over 700,000 deaths annually. Mosquito-borne arbovirus outbreaks can range from global to regional. In the East African Community (EAC) region, these viruses have caused a series of emerging and reemerging infectious disease outbreaks. Member states in the EAC share a lot in common including regional trade and transport, some of the factors highlighted to be the cause of mosquito-borne arbovirus disease outbreaks worldwide. In this review, characteristics of 24 mosquito-borne arboviruses indigenous to the EAC are reviewed, including lesser or poorly understood viruses, like Batai virus (BATV) and Ndumu virus (NDUV), which may escape their origins under perfect conditions to establish a foothold in new geographical locations. Factors that may influence the future spread of these viruses within the EAC are addressed. With the continued development observed in the EAC, strategies should be developed by the Community in improving mosquito and mosquito-borne arbovirus surveillance to prevent future outbreaks.

Arboviruses in the East African Community partner states: a review of medically important mosquito-borne Arboviruses.

Phage stability is important for the successful application of bacteriophages as alternative antibacterial agents. Considering that temperature is a critical factor in phage stability, this study aimed to explore the possibility of improving long-term phage stability through adaptive evolution to elevated temperature. Evolution of three wild-type ancestral phages ( phage Wc4 and  phages CX5 and P-PSG-11) was induced by subjecting the phages to heat treatment at 60 °C for five cycles. The adapted phages showed better stability than the wild-type ancestral phages when subjected to heat treatment at 60 °C for 1 h and after 60 days of storage at 37 °C. However, the adapted phages could not withstand thermal treatment at 70 °C for 1 h. The infectivity and the lytic properties of the phages were not changed by the evolution process. Whole-genome sequencing revealed that single substitutions in the tail tubular proteins were the only changes observed in the genomes of the adapted phages. This study demonstrates that adaptive evolution could be used as a general method for enhancing the thermal stability of phages without affecting their lytic activity. Sequencing results showed that bacteriophages may exist as a population with minor heterogeneous mutants, which might be important to understand the ecology of phages in different environments.

Application of Adaptive Evolution to Improve the Stability of Bacteriophages during Storage.

Droplet digital PCR (ddPCR) is a third generation of PCR that was recently developed to overcome the challenges of real-time fluorescence-based quantitative PCR (qPCR) in absolute quantification of pathogens. Few studies have been done on tuberculosis (TB) detection and quantification using ddPCR despite its many advantages over qPCR. From the few studies, none explores a single dye duplex assay for the detection and quantification of TB. In this study, steps toward developing and evaluating a duplex single dye (FAM) assay for detecting two targets ( and  are clearly described using simplex and duplex experiments. To achieve this, various parameters are investigated, including annealing temperature, primer and probe concentration, sensitivity and specificity, sample concentration, and inter/intra-assay variability. From the results, primer and probe concentration, annealing temperature, and sample concentration have an effect on the position and separation of droplets in both simplex and duplex assays. The copies of target genes in a duplex assay can be estimated accurately using the threshold tool with little inter-assay (CV <1%) and intra-assay (CV <6%) variability when compared to simplex assays. The ddPCR assay specificity and sensitivity are both 100% when compared to qPCR. This work shows steps toward the detection and quantification of two targets in a single channel, enabling higher multiplexing to include more targets in future works.

Development and Evaluation of a Single Dye Duplex Droplet Digital PCR Assay for the Rapid Detection and Quantification of .

SARS-CoV-2 Does Not Replicate in Aedes Mosquito Cells nor Present in Field-Caught Mosquitoes from Wuhan.

Zika virus (ZIKV) is an emerging arthropod-borne flavivirus that, upon infection, results in teratogenic effects and neurological disorders. ZIKV infections pose serious global public health concerns, prompting scientists to increase research on antivirals and vaccines against the virus. These efforts are still ongoing as the pathogenesis and immune evasion mechanisms of ZIKV have not yet been fully elaborated. Currently, no specific vaccines or drugs have been approved for ZIKV; however, some are undergoing clinical trials. Notably, several strategies have been used to develop antivirals, including drugs that target viral and host proteins. Additionally, drug repurposing is preferred since it is less costly and takes less time than other strategies because the drugs used have already been approved for human use. Likewise, different platforms have been evaluated for the design of vaccines, including DNA, mRNA, peptide, protein, viral vectors, virus-like particles (VLPSs), inactivated-virus, and live-attenuated virus vaccines. These vaccines have been shown to induce specific humoral and cellular immune responses and reduce viremia and viral RNA both  and . Importantly, most of these vaccines have entered clinical trials. Understanding the viral disease mechanism will provide better strategies for developing therapeutic agents against ZIKV. This review provides a comprehensive summary of the viral pathogenesis of ZIKV and current advancements in the development of vaccines and drugs against this virus.

Zika virus pathogenesis and current therapeutic advances.

: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard 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. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening.: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system.: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples 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  while another testing drug could not.: 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.

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

 are a group of viruses with significant public and veterinary health importance. These viruses are mainly transmitted through mosquito-, midge-, and tick-vectors, and are endemic to various regions of the world. Ebinur Lake virus (EBIV), a newly identified member of , was isolated from  mosquitoes in Northwest China. In the present study, we aimed to characterize the pathogenesis and host immune responses of EBIV in BALB/c mice, as an animal model. Herein, we determined that BALB/c mice are highly susceptible to EBIV infection. The infected mice exhibited evident clinical signs including weight loss, mild encephalitis, and death. High mortality of mice was observed even with inoculation of one plaque-forming unit (PFU) of EBIV, and the infected mice succumbed to death within 5-9 days. After EBIV challenge, rapid viremic dissemination was detected in the peripheral tissues and the central nervous system, with prominent histopathologic changes observed in liver, spleen, thymus, and brain. Blood constituents' analysis of EBIV infected mice exhibited leukopenia, thrombocytopenia, and significantly elevated ALT, LDH-L, and CK. Further, EBIV infection induced obvious cytokines changes in serum, spleen, and brain in mice. Collectively, our data describe the first study that systematically examines the pathogenesis of EBIV and induced immune response in an immunocompetent standard mouse model, expanding our knowledge of this virus, which may pose a threat to One Health.

Raphael Nyaruaba

Key Laboratory of Special Pathogens and Biosafety,
Center for Biosafety Mega-Science,
Wuhan Institute of Virology,
Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology

Chinese Academy of Sciences

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification