SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast ...and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.
Background. There is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and a surge of SARS-CoV-2 cases, especially in limited-resource ...settings. Therefore, the combination of an optimal assay kit is necessary. Methods. We compared the ability to screen SARS-CoV-2 among three primer-probe sets in two different master mixes, Invitrogen™ SuperScript™ III One-Step RT-PCR and LightCycler Multiplex RNA Virus Master. Results. The assay with TIB-Molbiol, IDT, and Phu Sa sets for LightCycler Multiplex RNA Virus Master or Invitrogen™ SuperScript™ III One-Step RT-PCR showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. R squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. Conclusions. Our findings indicated that TIB-Molbiol, IDT, and Phu Sa primer-probe sets could be beneficial for the laboratory screening of SARS-CoV-2 by RT-qPCR assay of E gene. There is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for COVID-19.
Abstract
Background
Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and ...management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests.
Methods
We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT.
Results
Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation.
Conclusion
Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.
Over the last decade, nucleic acid amplification tests (NAATs) including polymerase chain reaction (PCR) were an indispensable methodology for diagnosing cancers, viral and bacterial infections owing ...to their high sensitivity and specificity. Because the NAATs can recognize and discriminate even a few copies of nucleic acid (NA) and species-specific NA sequences, NAATs have become the gold standard in a wide range of applications. However, limitations of NAAT approaches have recently become more apparent by reason of their lengthy run time, large reaction volume, and complex protocol. To meet the current demands of clinicians and biomedical researchers, new NAATs have developed to achieve ultrafast sample-to-answer protocols for the point-of-care testing (POCT). In this review, ultrafast NA-POCT platforms are discussed, outlining their NA amplification principles as well as delineating recent advances in ultrafast NAAT applications. The main focus is to provide an overview of NA-POCT platforms in regard to sample preparation of NA, NA amplification, NA detection process, interpretation of the analysis, and evaluation of the platform design. Increasing importance will be given to innovative, ultrafast amplification methods and tools which incorporate artificial intelligence (AI)-associated data analysis processes and mobile-healthcare networks. The future prospects of NA POCT platforms are promising as they allow absolute quantitation of NA in individuals which is essential to precision medicine.
•The nucleic acid amplification test (NAAT) platform includes the sample preparation, NA amplification, NA detection process, and interpretation of the analysis.•The innovative ultrafast NA amplification including light-, radio frequency-, convective flow- and resistive-assisted thermal cycling was reviewed.•The NAATs can be connected with mobile-healthcare and artificial intelligence for absolute quantitation of NA in individuals.•The ultrafast NAATs can make impacts in precision medicine, personalized and preventive medicine.
Severe acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. Multiplex real time RT-PCR helps in simultaneous ...detection of multiple viruses saving cost, time and labour. Commercially available multiplex real time RT-PCR kits are very expensive. Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD).
Nasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples.
Custom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively. The concordance between the custom assay and the FTD assay was 100 % for HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A(H1N1)pdm09 and 94.66 - 99.71 % for the remaining viruses; Flu B (99.71 %), HRV (99.71 %), HPIV-3 (98.87 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), EV (98.31 %), HCoV HKU1 (99.71 %), HAdV (99.71 %). Major discrepancy was observed for RSV A/B, which was over detected in 18 samples by the custom assay as compared to the FTD assay. The custom assay was much cheaper than the FTD assay and the time taken was only 29 min more.
The custom primer and probe mix was found to be comparable to the FTD assay with good concordance but was much cheaper and the time taken for reporting was only 29 min more. The low cost custom multiplex RT-PCR can be a useful alternative to the costly FTD kit for rapid identification of viral aetiology in resource limited settings.
Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and ...in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described.
This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence.
In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.
Bovine respiratory disease complex (BRDC) is one of the most significant diseases of cattle. Bacterial pathogens involved in BRDC include Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, ...and Pasteurella multocida. We developed and evaluated a multiplexed real-time hydrolysis probe (rtPCR) assay using block-based Peltier and rotary-based thermocycling on lung tissue, nasal swabs, and deep nasopharyngeal swabs. The rtPCR results were compared to culture or a gel-based M. bovis PCR using statistical analysis to determine optimum quantification cycle (Cq) cutoffs to maximize agreement. The limits of detection were 1.2–12 CFU/reaction for each pathogen. M. haemolytica was the most prevalent organism detected by rtPCR, and was most frequently found with P. multocida. The rtPCR assay enabled enhanced levels of detection over culture for all pathogens on both thermocycling platforms. The rotary-based thermocycler had significantly lower Cq cutoffs (35.2 vs. 39.7), which maximized agreement with gold standard culture or gel-based PCR results following receiver operating characteristic analysis to maximize sensitivity (Se) and specificity (Sp). However, overall assay Se and Sp were similar on both platforms (80.5% Se, 88.8% Sp vs. 80.1% Se, 88.3% Sp). Implementation of these tests could enhance the detection of these pathogens, and with high-throughput workflows could reduce assay time and provide more rapid results. The assays may be especially valuable in identifying coinfections, given that many more antemortem samples tested in our study were positive for 2 or more pathogens by rtPCR (n = 125) than were detected using culture alone (n = 25).
An ideal test for COVID-19 would combine the sensitivity of laboratory-based PCR with the speed and ease of use of point-of-care (POC) or home-based rapid antigen testing. We evaluated clinical ...performance of the Diagnostic Analyzer for Selective Hybridization (DASH) SARS-CoV-2 POC rapid PCR test. We conducted a cross-sectional study of adults with and without symptoms of COVID-19 at four clinical sites where we collected two bilateral anterior nasal swabs and information on COVID-19 symptoms, vaccination, and exposure. One swab was tested with the DASH SARS-CoV-2 POC PCR and the second in a central laboratory using Cepheid Xpert Xpress SARS-CoV-2 PCR. We assessed test concordance and calculated sensitivity, specificity, negative and positive predictive values using Xpert as the "gold standard". We enrolled 315 and analyzed 313 participants with median age 42 years; 65% were female, 62% symptomatic, 75% had received greater than or equal to2 doses of mRNA COVID-19 vaccine, and 16% currently SARS-CoV-2 positive. There were concordant results for 307 tests indicating an overall agreement for DASH of 0.98 95% CI 0.96, 0.99 compared to Xpert. DASH performed at 0.96 95% CI 0.86, 1.00 sensitivity and 0.98 95% CI 0.96, 1.00 specificity, with a positive predictive value of 0.85 95% CI 0.73, 0.96 and negative predictive value of 0.996 95% CI 0.99, 1.00. The six discordant tests between DASH and Xpert all had high Ct values (>30) on the respective positive assay. DASH and Xpert Ct values were highly correlated (R = 0.89 95% CI 0.81, 0.94). DASH POC SARS-CoV-2 PCR was accurate, easy to use, and provided fast results (approximately 15 minutes) in real-life clinical settings with an overall performance similar to an EUA-approved laboratory-based PCR.
OBJECTIVE: To determine whether microRNAs are differentially expressed in men with normal versus impaired spermatogenesis, and to find a biomarker for accurate diagnosis of male infertility. DESIGN: ...Microarray with real-time polymerase chain reaction (RT-PCR) validation. SETTING: University research and clinical institutes. PATIENT(S): Male partner of selected couples (n = 27) who were undergoing assisted reproduction techniques for infertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Statistically significantly altered microRNA expression profiles in normozoospermic versus asthenozoospermic and oligoasthenozoospermic men. RESULT(S): There were 50 miRNAs up-regulated and 27 miRNAs down-regulated in asthenozoospermic males. In oligoasthenozoospermic males, 42 miRNAs were up-regulated and 44 miRNAs down-regulated when compared with normozoospermic males. The miRNAs that exhibited the highest fold changes and area under the receiver operating characteristic curve were miR-34b, miR-122, and miR-1973 in samples from asthenozoospermic men and miR-34b, miR-34b*, miR-15b, miR-34c-5p, miR-122, miR-449a, miR-1973, miR-16, and miR-19a in samples from oligoasthenozoospermic men. Furthermore, quantitative RT-PCR assays on specific miRNAs, including miR-141, miR-200a, miR-122, miR-34b, miR-34c-5p, and miR-16, yielded results that were largely consistent with the microarray data. CONCLUSION(S): Our results reveal an extended number of miRNAs that were differentially expressed in asthenozoospermic and oligoasthenozoospermic males compared with normozoospermic males. These data provide evidence for analysis of miRNA profiles as a future diagnosing tool for male infertility.
Pigs are frequently infected with porcine cytomegalovirus (PCMV). Infected adult animals may not present with symptoms of disease, and the virus remains latent. However, the virus may be transmitted ...to human recipients receiving pig transplants. Recently, it was shown that pig-to-non-human-primate xenotransplantations showed 2 to 3 times lower transplant survival when the donor pig was infected with PCMV. Therefore, highly sensitive methods are required to select virus-free pigs and to examine xenotransplants. Seven previously established PCR detection systems targeting the DNA polymerase gene of PCMV were examined by comparison of thermodynamic parameters of oligonucleotides, and new diagnostic nested PCR and real-time PCR systems with improved parameters and high sensitivity were established. The detection limit of conventional PCR was estimated to be 15 copies, and that of the nested PCR was 5 copies. The sensitivity of the real-time PCR with a TaqMan probe was two copies. An equal efficiency of the newly established detection systems was shown by parallel testing of DNA from sera and blood of six pigs, identifying the same animals as PCMV infected. These new diagnostic PCR systems will improve the detection of PCMV and therefore increase the safety of porcine xenotransplants.
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