Detection Of Viral Nucleic Acids Research Articles

A Novel High-Throughput Sample-in-Result-Out Device for the Rapid Detection of Viral Nucleic Acids

Clustered regularly interspaced short palindromic repeats (CRISPR) molecular diagnostic technology is one of the most reliable diagnostic tools for infectious diseases due to its short reaction time, high sensitivity, and excellent specificity. However, compared with fluorescent polymerase chain reaction (PCR) technology, CRISPR molecular diagnostic technology lacks high-throughput automated instrumentation and standardized detection reagents for high sensitivity, limiting its large-scale clinical application. In this study, a high-throughput automated device was developed by combining reagent lyophilization, extraction-free technology, and a one-pot consumable system. This innovative approach enabled the rapid sample-in-result-out detection of 48 samples in 25 min and demonstrated high sensitivity and specificity for the qualitative analysis of clinical samples. The obtained results show that the detection limit of the designed system for African swine fever virus (ASFV) is 0.5 copies/μL. As a proof concept, a single-tube dual-target nucleic acid detection method was developed, achieving a detection limit of 5 copies/μL for the ORF1ab and N genes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within 45 min. The method is highly specific, reliable, and stable, providing a feasible solution for the clinical application of CRISPR nucleic acid detection technology.

Quantum Dot Reporters Designed for CRISPR-Based Detection of Viral Nucleic Acids.

Diagnostic methods based on CRISPR technology have shown great potential due to their highly specific, efficient, and sensitive detection capabilities. Although the majority of the current studies rely on fluorescent dye-quencher reporters, the limitations of fluorescent dyes, such as poor photostability and small Stokes shifts, urgently necessitate the optimization of reporters. In this study, we developed innovative quantum dot (QD) reporters for the CRISPR/Cas systems, which not only leveraged the advantages of high photoluminescence quantum yield and large Stokes shifts of QDs but were also easily synthesized through a simple one-step hydrothermal method. Based on the trans-cleavage characteristics of Cas12a and Cas13a, two types of QD reporters were designed, the short DNA strand and the hybridization-based QD reporters, achieving the detection of DNA and RNA at the pM level, respectively, and validating the performance in the analysis of clinical samples. Furthermore, based on the unique property of QDs that allowed multicolor emission under one excitation, the application potential for simultaneous detection of diseases was further investigated. Taken together, this work proposed novel QD reporters that could be applied to the various CRISPR/Cas systems, providing a new toolbox to expand the diagnosis of bioanalytical and biomedical fields.

Analysis of serum IgM antibody and viral nucleic acid test results in measles and rubella cases in China from 2014 to 2023

Objective: To analyze the results of serum IgM antibody and viral nucleic acid testing in measles and rubella cases in China from 2014 to 2023. Methods: Surveillance data on measles and rubella during 2014-2023 were obtained from the Chinese Disease Prevention and Control Information System, and cases that underwent measles/rubella IgM antibody testing and viral nucleic acid testing were included in the study. Information on the number of cases, vaccination status, and laboratory test results was collected, and laboratory test results were compared among cases with different times of rash or onset and different doses of vaccination. Results: From 2014 to 2023, the total number of measles and rubella surveillance cases was 581 746, and the number of measles and rubella cases that underwent both IgM antibody and viral nucleic acid testing was 39 124 and 21 766, respectively, with a double-positive rate of IgM antibody and viral nucleic acid testing of 63.73% (for measles) and 41.68% (for rubella). The single-positive rate for IgM antibody was 21.12% (for measles) and 27.15% (for rubella). The single-positive rate for viral nucleic acid tests was 13.84% (for measles) and 28.19% (for rubella). According to different days of rash or onset (0-3, 4-5, 6-10 and >10), samples of 24 629 (62.95%) measles cases and 15 785 (72.52%) rubella cases were from within 3 days of rash or onset, and the single positive rate of viral nucleic acid detection was 16.90% (for measles) and 32.07% (for rubella). The single positive rate of IgM antibody increased gradually with the time of onset (measles: χ2trend=314.098, P<0.001, rubella: χ2trend=763.846, P<0.001), and reached 31.66% (for measles) and 53.12% (for rubella) after 10 days of rash or onset. Depending on the number of vaccination doses (1, 2,≥3), the single positive rate of viral nucleic acid detection ranged from 12.93% to 20.29% (for measles) and from 25.30% to 27.88% (for rubella). The single positive rate of IgM antibody detection ranged from 36.89% to 47.47% (for measles) and from 22.81 to 41.15% (for rubella). Conclusions: Combined testing of serum IgM antibody and viral nucleic acids could facilitate laboratory confirmation of measles and rubella cases and was also important for measles and rubella elimination efforts.

An Fe3O4-Au heterodimer nanoparticle-based lateral flow assay for rapid and simultaneous detection of multiple influenza virus nucleic acids.

Sensitive, convenient and rapid detection and subtyping of influenza viruses are crucial for timely treatment and management of infected people. Compared with antigen detection, nucleic acid detection has higher specificity and can shorten the detection window. Hence, in this work, we improved the lateral flow assay (LFA, one of the most promising user-friendly and on-site methods) to achieve detection and subtyping of H1N1, H3N2 and H9N2 influenza virus nucleic acids. Firstly, the antigen-antibody recognition mode was transformed into a nucleic acid hybridization reaction. Secondly, Fe3O4-Au heterodimer nanoparticles were prepared to replace frequently used Au nanoparticles to obtain better coloration. Thirdly, four lines were arranged on the LFA strip, which were three test (T) lines and one control (C) line. Three T lines were respectively sprayed by the DNA sequences complementary to one end of H1N1, H3N2 and H9N2 influenza virus nucleic acids, while Fe3O4-Au nanoparticles were respectively coupled with the DNA sequences complementary to the other end of H1N1, H3N2 and H9N2 nucleic acids to construct three kinds of probes. The C line was sprayed by the complementary sequences to the DNAs on all three kinds of probes. In the detection, by hybridization reaction, the probes were combined with their target nucleic acids which were captured by the corresponding T lines to form color bands. Finally, according to the position of the color bands and their grey intensity, simultaneous qualitative and semi-quantitative detection of the three influenza virus nucleic acids was realized. The detection results showed that this multi-channel LFA had good specificity, and there was no significant cross reactivity among the three subtypes of influenza viruses. The simultaneous detection achieved comparable detection limits with individual detections. Therefore, this multi-channel LFA had good application potential for sensitive and rapid detection and subtyping of influenza viruses.

Analysis of seasonal H3N2 influenza virus epidemic characteristics and whole genome features in Jining City from 2018 to 2023.

Seasonal H3N2 influenza virus, known for its rapid evolution, poses a serious threat to human health. This study focuses on analyzing the influenza virus trends in Jining City (2018-2023) and understanding the evolving nature of H3N2 strains. Data on influenza-like cases were gathered from Jining City's sentinel hospitals: Jining First People's Hospital and Rencheng Maternal and Child Health Hospital, using the Chinese Influenza Surveillance Information System. Over the period from 2018 to 2023, 7844 throat swab specimens were assessed using real-time fluorescence quantitative PCR for influenza virus nucleic acid detection. For cases positive for seasonal H3N2 influenza virus, virus isolation was followed by whole genome sequencing. Evolutionary trees were built for the eight gene segments, and protein variation analysis was performed. From 2018 to 2023, influenza-like cases in Jining City represented 6.99% (237 299/3 397 247) of outpatient visits, peaking in December and January. Influenza virus was detected in 15.67% (1229/7844) of cases, primarily from December to February. Notably, no cases were found in the 2020-2021 season. Full genome sequencing was conducted on 70 seasonal H3N2 strains, revealing distinct evolutionary branches across seasons. Significant antigenic site variations in the HA protein were noted. No resistance mutations to inhibitors were found, but some strains exhibited mutations in PA, NS1, PA-X, and PB1-F2. Influenza trends in Jining City saw significant shifts in the 2020-2021 and 2022-2023 seasons. Seasonal H3N2 exhibited rapid evolution. Sustained vigilance is imperative for vaccine updates and antiviral selection.

RNA extraction-free reduced graphene oxide-based RT-LAMP fluorescence assay for highly sensitive SARS-CoV-2 detection

Infectious diseases have always been a seriously endanger for human life and health. A rapid, accurate and ultra-sensitive virus nucleic acid detection is still a challenge to deal with infectious diseases. Here, a RNA extraction-free reduced graphene oxide-based reverse transcription-loop-mediated isothermal amplification (EF-G-RT-LAMP) fluorescence assay was developed to achieve high-throughput, rapid and ultra-sensitive SARS-CoV-2 RNA detection. The whole detection process only took ∼36 min. The EF-G-RT-LAMP assay achieves a detection limit of 0.6 copies μL−1 with a wide dynamic range of aM-pM. A large number (up to 384) of samples can be detected simultaneously. Simulated detection of the COVID-19 pseudovirus and clinical samples in nasopharyngeal swabs demonstrated a high-throughput, rapid and ultra-sensitive practical detection capability of the EF-G-RT-LAMP assay. The results proved that the assay would be used as a rapid, easy-to-implement approach for epidemiologic diagnosis and could be extended to other nucleic acid detections.

Logistic regression analysis of the value of biomarkers, clinical symptoms, and imaging examinations in COVID-19 for SARS-CoV-2 nucleic acid detection.

The detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) nucleic acid detection provides a direct basis for diagnosing Coronavirus Disease 2019. However, nucleic acid test false-negative results are common in practice and may lead to missed diagnosis. Certain biomarkers, clinical symptoms, and imaging examinations are related to SARS-CoV-2 nucleic acid detection and potential predictors. We examined nucleic acid test results, biomarkers, clinical symptoms, and imaging examination data for 116 confirmed cases and asymptomatic infections in Zhuhai, China. Patients were divided into nucleic acid-positive and -false-negative groups. Predictive values of biomarkers, symptoms, and imaging for the nucleic acid-positive rate were calculated by Least Absolute Shrinkage and Selection Operators regression analysis and binary logistic regression analysis, and areas under the curve of these indicators were calculated. Hemoglobin (OR = 1.018, 95% CI: 1.006-1.030; P = .004) was higher in the respiratory tract-positive group than the nucleic acid-negative group, but platelets (OR = 0.996, 95% CI: 0.993-0.999; P = .021) and eosinophils (OR = 0.013, 95% CI: 0.001-0.253; P = .004) were lower; areas under the curve were 0.563, 0.614, and 0.642, respectively. Some biomarkers can predict SARS-CoV-2 viral nucleic acid detection rates in Coronavirus Disease 2019 and are potential auxiliary diagnostic tests.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Inactivation of highly transmissible livestock and avian viruses including influenza A and Newcastle disease virus for molecular diagnostics.

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection while maintaining the nucleic acid quality required for diagnostic PCR testing. This inactivation method is required to alleviate concerns about transmission potential, minimize shipping complications and cost, and enable testing in lower containment laboratories, thereby enhancing disease diagnostics through improved turn-around time. This study evaluated a panel of 10 surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial PrimeStore® molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99%, as determined by infectivity and serial passage assays. However, the detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and the shipment of infectious substances.

International review of blood donation nucleic acid amplification testing.

Nucleic acid amplification testing (NAT), in blood services context, is used for the detection of viral and parasite nucleic acids to reduce transfusion-transmitted infections. This project reviewed NAT for screening blood donations globally. A survey on NAT usage, developed by the International Society of Blood Transfusion Working Party on Transfusion-transmitted Infectious Diseases (ISBT WP-TTID), was distributed through ISBT WP-TTID members. Data were analysed using descriptive statistics. Forty-three responses were received from 32 countries. Increased adoption of blood donation viral screening by NAT was observed over the past decade. NAT-positive donations were detected for all viruses tested in 2019 (proportion of donations positive by NAT were 0.0099% for human immunodeficiency virus [HIV], 0.0063% for hepatitis C virus [HCV], 0.0247% for hepatitis B virus [HBV], 0.0323% for hepatitis E virus [HEV], 0.0014% for West Nile virus [WNV] and 0.00005% for Zika virus [ZIKV]). Globally, over 3100 NAT-positive donations were identified as NAT yield or solely by NAT in 2019 and over 22,000 since the introduction of NAT, with HBV accounting for over half. NAT-positivity rate was higher in first-time donors for all viruses tested except WNV. During 2019, a small number of participants performed NAT for parasites (Trypanosoma cruzi, Babesia spp., Plasmodium spp.). This survey captures current use of blood donation NAT globally. There has been increased NAT usage over the last decade. It is clear that NAT contributes to improving blood transfusion safety globally; however, there is a need to overcome economic barriers for regions/countries not performing NAT.

Development of a highly sensitive TaqMan method based on multi-probe strategy: its application in ASFV detection.

The establishment of high sensitive detection method for various pathogenic microorganisms remains constantly concerned. In the present study, multi-probe strategy was first systematically investigated followed by establishing a highly sensitive TaqMan real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus (ASFV). Briefly, four probes based on the B646L gene of ASFV were designed and the effects of different combinations of the probes in a single TaqMan qPCR assay on the detection sensitivity were investigated. As less as 0.5-5 copies/μl of the ASFV gene was detected by the established TaqMan qPCR assay. Furthermore, plasmid harboring the B646L in water samples could be concentrated 1000 times by ultrafiltration to enable a highly sensitive detection of trace viral nucleic acids. Moreover, no cross-reactivity was observed with other common clinical swine viruses such as PCV2, PCV3, PCV4, PEDV, PDCoV, CSFV, PRRSV, and PRV. When detecting 173 clinical porcine serum samples, the coincidence rate between the developed method and WOAH (World Organization of Animal Health) recommended method was 100%. This study might provide an integrated strategy to achieve higher detection sensitivity of trace pathogenic microorganisms and applicably sensitive TaqMan-based qPCR assays.

Advances in rapid point-of-care virus testing.

Outbreaks of viral diseases seriously jeopardize people's health and cause huge economic losses. At the same time, virology provides a new perspective for biology, molecular biology and cancer research, and it is important to study the discovered viruses with potential applications. Therefore, the development of immediate and rapid viral detection methods for the prevention and treatment of viral diseases as well as the study of viruses has attracted extensive attention from scientists. With the continuous progress of science and technology, especially in the field of bioanalysis, a series of new detection techniques have been applied to the on-site rapid detection of viruses, which has become a powerful approach for human beings to fight against viruses. In this paper, the latest research progress of rapid point-of-care detection of viral nucleic acids, antigens and antibodies is presented. In addition, the advantages and disadvantages of these technologies are discussed from the perspective of practical application requirements. Finally, the problems and challenges faced by rapid viral detection methods and their development prospects are discussed.

Indoor Air Sampling for Detection of Viral Nucleic Acids

Indoor Air Sampling for Detection of Viral Nucleic Acids

Nucleic acid amplification testing (NAT) impact on blood safety compared to Immunoassays in blood banks: A Review

Blood transfusions are fundamental to clinical procedures; however, many people worldwide cannot access safe blood. Blood product safety must be essential in each country’s national health policies. Several aspects of the blood donation process are carefully performed, including laboratory testing comprising blood type determination, antigen-antibody analyses, and nucleic acid amplification testing (NAT); however, NAT is not mandatory in all countries. The traditional screening method is based on antigen-antibody binding techniques, such as ELISA (enzyme-linked immunosorbent assay), with high sensitivity and specificity. Nevertheless, these methods have a seroconversion window period (WP), in which antigen-antibody testing cannot detect the pathogen and has not caused any symptoms yet. NAT is a sensitive molecular method based on viral nucleic acid amplification and detection. Moreover, its use in blood banks is increasing worldwide because it narrows the window period. For example, Huang et al. in 2017 reported the detection of 22 samples reactive only by nucleic acid testing for either HIV, HBV, or HCV compared with ELISA. The present article shows how blood safety has improved by implementing NAT as a routine method for viral nucleic acid detection, highlighting the importance of this technique as evidenced by the findings presented herein. Moreover, these results are highly significant, demonstrating the relevance of NAT and advocating for its application on a global scale in blood management protocols. This development could be particularly beneficial for regions with a high viral infection prevalence, including many countries. Keywords: Nucleic acid amplification, Immunoassay, viral infection, blood bank.

A Biotechnological Review on Patents Applied to Rubella Diagnosis.

Rubella, caused by the Rubella virus (RV), is considered a mild self-limited illness. However, RV has teratogenic potential. Laboratory investigation plays an important role in both diagnosis and surveillance of the disease. The main methods for diagnosing Rubella are serological assays for the detection of specific IgM and molecular assays for detecting viral RNA. However, some laboratories perform IgG avidity testing, virus isolation and analysis of genetic sequence as tools to help Rubella eradication. The importance of the diagnosis of Rubella involves the appropriate treatment of the disease, because the Rubella clinical symptoms may be similar to those of other diseases, and the population monitoring to avoid new emergent cases. This study addresses different methods of diagnosing Rubella and contributes as a source of knowledge to assist health systems in controlling the disease. The main objective of this study was to review the available patents regarding Rubella diagnosis published in intellectual property databases, and provides an overview of the technologies available for the diagnosis of Rubella. The search strategy was based on the keywords searched separately or together using a Boolean operator either in the patent title or abstract the time interval was restricted to patents filed or granted from January 2009 until February 2022. The database used was Google Patents. This study analyzed 24 patent documents regarding strategies for the diagnosis of Rubella. Of these, 15 patents disclose strategies for detecting Rubella antibodies, 7 patents the detection of Rubella virus nucleic acid, and 2 patents the production of antibodies applied in Rubella diagnosis. Rubella is still a public health problem in some countries, mainly those in development, especially due to congenital Rubella syndrome, which can cause malformation or fetal death. However, its diagnosis is challenging, due to similarity of symptoms with other diseases, and for this reason, laboratory diagnosis is essential. Studies like this encourage researchers and governments to invest in research to continue the development of new products, using different areas of biotechnology, to solve society's problems, especially diseases that have an impact on global health, such as Rubella.

Rift valley fever outbreak in Sembabule District, Uganda, December 2020

BackgroundRift Valley Fever (RVF) is a viral zoonosis that can cause severe haemorrhagic fevers in humans and high mortality rates and abortions in livestock. On 10 December 2020, the Uganda Ministry of Health was notified of the death of a 25-year-old male who tested RVF-positive by reverse-transcription polymerase chain reaction (RT-PCR) at the Uganda Virus Research Institute. We investigated to determine the scope of the outbreak, identify exposure factors, and institute control measures.MethodsA suspected case was acute-onset fever (or axillary temperature > 37.5 °C) and ≥ 2 of: headache, muscle or joint pain, unexpected bleeding, and any gastroenteritis symptom in a resident of Sembabule District from 1 November to 31 December 2020. A confirmed case was the detection of RVF virus nucleic acid by RT-PCR or serum IgM antibodies detected by enzyme-linked immunosorbent assay (ELISA). A suspected animal case was livestock (cattle, sheep, goats) with any history of abortion. A confirmed animal case was the detection of anti-RVF IgM antibodies by ELISA. We took blood samples from herdsmen who worked with the index case for RVF testing and conducted interviews to understand more about exposures and clinical characteristics. We reviewed medical records and conducted an active community search to identify additional suspects. Blood samples from animals on the index case’s farm and two neighbouring farms were taken for RVF testing.ResultsThe index case regularly drank raw cow milk. None of the seven herdsmen who worked with him nor his brother’s wife had symptoms; however, a blood sample from one herdsman was positive for anti-RVF-specific IgM and IgG. Neither the index case nor the additional confirmed case-patient slaughtered or butchered any sick/dead animals nor handled abortus; however, some of the other herdsmen did report high-risk exposures to animal body fluids and drinking raw milk. Among 55 animal samples collected (2 males and 53 females), 29 (53%) were positive for anti-RVF-IgG.ConclusionsTwo human RVF cases occurred in Sembabule District during December 2020, likely caused by close interaction between infected cattle and humans. A district-wide animal serosurvey, animal vaccination, and community education on infection prevention practices campaign could inform RVF exposures and reduce disease burden.

The establishment and application of a dual Nano-PCR detection method for feline calicivirus and feline herpesvirus type I.

Feline calicivirus (FCV) and Feline herpesvirus type I (FHV-I) are the main pathogens causing upper respiratory tract infections in cats, and some wild animals. These two viruses always coinfection and cause serious harm to pet industry and wild animals protection. Established a rapid and accurate differential diagnosis method is crucial for prevention and control of disease, however, the current main detection method for these two viruses, either is low sensitivity (immunochromatographic strip), or is time-consuming and cannot differential diagnosis (conventional single PCR). Nanoparticle-assisted polymerase chain reaction (Nano-PCR) is a recently developed technique for rapid detection method of virus and bacteria. In this study, we described a dual Nano-PCR assay through combining the nanotechnology and PCR technology, which for the clinical simultaneous detection of FCV and FHV-I and differential diagnosis of upper respiratory tract infections in cats or other animals. Under optimized conditions, the optimal annealing temperature for dual Nano-PCR was 51.5°C, and specificity test results showed it had no cross reactivity to related virus, such as feline panleukopenia virus (FPV), feline Infectious peritonitis virus (FIPV) and rabies virus (RABV). Furthermore, the detection limit of dual Nano-PCR for FCV and FHV-I both were 1 × 10-8 ng/μL, convert to number of copies of virus DNA was 6.22 × 103copies/μL (FCV) and 2.81 × 103copies/μL (FHV-I), respectively. The dual Nano-PCR detected result of 52 cat clinical samples, including ocular, nasal and faecal swabs, and (3 FCV-positive samples), was consistent with ordinary PCR and the clinical detection results. The dual Nano-PCR method established in this study with strong specificity and high sensitivity can be used for virus nucleic acid (FCV and FHV-I) detection of clinical samples of feline upper respiratory tract infections feline calicivirus and feline herpesvirus while providing support for the early diagnosis of cats that infected by FCV and FHV-I.

Digital CRISPR/Cas13a-RPA based assay for rapid and sensitive detection of JC virus in renal transplant patients

JC viruses (JCVs), members of the Polyomaviruses family, are a type of circular double-stranded DNA (dsDNA) viruses and can lead to opportunistic infections. Infection of JCVs in kidney transplant patients can lead to polyomavirus-associated nephropathy. Thus, early detection and quantification of JC virus in urine or blood will effectively improve the survival rate of kidney transplant patients and reduce the mortality of patients. At present, PCR has been used as a routine method for JC virus detection in clinical practice. However, PCR cannot achieve absolute quantitative detection of viral nucleic acid. rendering it unsuitable for certain clinical applications with specific requirements. Herein, we proposed a JC virus nucleic acid testing platform, named MdCaR, which couples a microfluidic droplet device with CRISPR-Cas13a and recombinase polymerase amplification. MdCaR can quantitatively detect JC virus through digital droplet. The detection limit achieves 1 copy/mL, and the assay takes only 40 min. Twelve renal transplant patients and 20 control subjects’ specimens were analyzed. MdCaR conveyed a good performance in testing these samples, especially for detecting the low viral load samples identified as negative by qPCR. Taken altogether, MdCaR could be regarded as a promising, rapid JCV detection strategy and widely applied for diagnosis for JCV infection.

Electrochemical Detection of Viral Nucleic Acids by DNA Nanolock-Based Porous Electrode Device.

Developing rapid, sensitive, and facile nucleic acid detection technologies is of paramount importance for preventing and controlling infectious diseases. Benefiting from the advantages such as rapid response, low cost, and simple operation, electrochemical impedance spectroscopy holds great promise for point-of-care nucleic acid detection. However, the sensitivity of electrochemical impedance spectroscopy for low molecular weight nucleic acids testing is still limited. This work presents a DNA nanolock-based porous electrode to improve the sensitivity of electrochemical impedance spectroscopy. Once the target nucleic acids are recognized by the DNA probes, the pore-attached DNA nanolock caused remarkable impedance amplification by blocking the nanopores. Taking SARS-CoV-2 nucleic acid as a model analyte, the detection limit of the porous electrode was as low as 0.03 fM for both SARS-CoV-2 RNA and DNA. The integration of a porous electrode with a wireless communicating unit generates a portable detection device that could be applied to direct SARS-CoV-2 nucleic acid testing in saliva samples. The portable device could effectively distinguish the COVID-19 positive and negative samples, showing a sensitivity of 100% and a specificity of 93%. Owing to its rapid, ultrasensitive, specific, and portable features, the as-designed DNA nanolock and porous electrode-based portable device holds great promise as a point-of-care platform for real-time screening of COVID-19 and other epidemics.

A fatal case of neonatal viral sepsis caused by human parainfluenza virus type 3

BackgroundSepsis is a systemic inflammatory response syndrome caused by severe infection in children, but cases of sepsis associated with human parainfluenza virus (HPIV) have been rarely reported in newborns.Case presentationWe report a case of HPIV-3 positive full-term newborn admitted to the Neonatal Intensive Care Unit of Beijing Children’s Hospital due to hematuria, gloomy spirit, inactivity and loss of appetite for 6 h. He had septic shock when he arrived the Accident & Emergency Department requiring immediate intubation and mechanical ventilation. Intravenous antibiotics were started. He had completely negative response to all anti-shock treatments including fluid resuscitation and vasopressor supports, and died 14 h later. Viral nucleic acid detection and metagenomic next-generation sequencing (mNGS) analyses of nasopharyngeal aspirate and blood specimens verified an HPIV-3 infection, with negative bacterial culture results. The HPIV-3 strain detected in this patient was subtyped as HPIV C3a, and two unreported amino acid mutations were found in the HN protein region.ConclusionThe patient had a severe infection associated with HPIV-3, which was the cause of sepsis and septic shock. This study showed the diagnostic value of mNGS in etiological diagnosis, especially in severe neonatal case.