Nucleic Amplification Research Articles

Versatile Luminol/Dissolved Oxygen/Fe@Fe2O3 Nanowire Ternary Electrochemiluminescence System Combined with Highly Efficient Strand Displacement Amplification for Ultrasensitive microRNA Detection.

Herein, a versatile ECL biosensor was fabricated for ultrasensitive detection of microRNA-21 (miRNA-21) from cancer cells based on a novel H2O2-free electrochemiluminescence (ECL) system (luminol/dissolved oxygen/Fe@Fe2O3 nanowires). Compared with the previously reported coreaction accelerator that needed a negative potential to produce reactive oxygen species (ROS), these newly discovered Fe@Fe2O3 nanowires could generate ROS in the detection solution immediately without the application of voltage, which narrowed down the detection potential range to avoid side reactions, favoring their practical application in biological systems. Especially, the Fe@Fe2O3 nanowires could produce H• for activating dissolved oxygen into ROS to improve the ECL intensity dramatically, which initiates a novel pathway to promote the generation of ROS for the ECL system. In addition, an original strand displacement amplification coupled with strand displacement reaction (SDA-SDR) was developed to improve the conversion efficiency of the target for sensitive detection of miRNA-21. By virtue of the SDR, a quadruple quenching effect was achieved through each output DNA strand of SDA; hence, the nucleic acid signal amplification efficiency was effectively enhanced. As expected, on account of the superb activation performance of Fe@Fe2O3 nanowires and the outstanding amplification efficiency of the SDR-SDA strategy, the fabricated ECL biosensor realized ultrasensitive detection of miRNA-21 with a detection limit down to 52.5 aM. The established ECL sensing platform ushered a new route for H2O2-free detection and a promising biomarker assay method for clinical diagnosis.

Modular DNA Circuits for Point-of-Care Colorimetric Assay of Infectious Pathogens.

Accurate, specific, and inexpensive detection of multiple infectious pathogens simultaneously is a significant goal for human health and safety. Herein we present a rationally designed modular DNA circuit for point-of-care (POC) detection of a variety of infectious pathogens based on nucleic acid isothermal amplification technology and DNAzyme-mediated colorimetric readout. A modular DNA circuit was constructed with a fixed module and a flexible module and was rationally designed according to genetic targets. On this basis, the platform could detect multiple genetic targets corresponding to infectious pathogens simultaneously. Signal amplification properties of the DNA circuit and the peroxidase-like DNAzyme enable the detection limits to reach the picomolar level. By urea treatment and magnetic separation, the fixed module can be reused at least five times, which makes this assay more economical and environmentally friendly. The detection of genetic infectious pathogens should be accomplished in 2 h with naked-eye observation and may provide an efficient tool for POC analysis of multiple infectious pathogens, especially in resource-poor areas.

A non-enzymatic, isothermal strand displacement and amplification assay for rapid detection of SARS-CoV-2 RNA

The current nucleic acid signal amplification methods for SARS-CoV-2 RNA detection heavily rely on the functions of biological enzymes which imposes stringent transportation and storage conditions, high cost and global supply shortages. Here, a non-enzymatic whole genome detection method based on a simple isothermal signal amplification approach is developed for rapid detection of SARS-CoV-2 RNA and potentially any types of nucleic acids regardless of their size. The assay, termed non-enzymatic isothermal strand displacement and amplification (NISDA), is able to quantify 10 RNA copies.µL−1. In 164 clinical oropharyngeal RNA samples, NISDA assay is 100 % specific, and it is 96.77% and 100% sensitive when setting up in the laboratory and hospital, respectively. The NISDA assay does not require RNA reverse-transcription step and is fast (<30 min), affordable, highly robust at room temperature (>1 month), isothermal (42 °C) and user-friendly, making it an excellent assay for broad-based testing.

A microfluidic surface-enhanced Raman scattering (SERS) sensor for microRNA in extracellular vesicles with nucleic acid-tyramine cascade amplification

Exosomal microRNA (miRNA) is an ideal candidate of noninvasive biomarker for the early diagnosis of cancer. Sensitive and accurate sensing of abnormal exosomal miRNA plays essential role for clinical promotion due to its close correlation with tumor proliferation and progression. Herein, a microfluidic surface-enhanced Raman scattering (SERS) sensor was proposed for an on-line detection of exosomal miRNA based on rolling circle amplification (RCA) and tyramine signal amplification (TSA) strategy. The microfluidic chip consists of a magnetic enrichment chamber, a serpentine fluidic mixer and a plasmonic SERS substrate functionalized with capture probes. The released miRNA activates the capture probe, triggers RCA reaction, and generates a large number of single-stranded DNA products to drive the catalysis of nanotags deposition via TSA, producing numerous “hot spots” to enhance the SERS signals. In merit of the microfluidics chip and nucleic acid-tyramine cascade amplification, the developed SERS sensor significantly improves the sensitivity for the exosomal miRNA assay, resulting in a limit of detection (LOD) as low as 1 pmol/L and can be successfully applied in the analysis of exosomes secreted from breast tumor cells, which demonstrates the potential utility in practical applications.

An enzyme-free three-dimensional DNA walker powered by catalytic hairpin assembly for H5N1 DNA ratiometric detection

The reliable and quantitative detection of virus DNA is highly desirable for the early diagnosis and clinical treatment of diseases. In this work, a three-dimensional (3D) DNA walker based on a non-enzyme-mediated nucleic acid cascade amplification reaction was constructed for the ratiometric detection of H5N1 DNA. When the DNA walker was developed by the surface modification of aminated silica microsphere (SiO2-NH2) with H1 hairpin structures were activated in the presence of target DNA, the fluorescence of the H1-conjugated Thioflavin T (ThT) fluorophore (495 nm) was turned on, while the fluorescence of 2-aminopurine (2-AP) (363 nm) conjugated to the H2 hairpin remained unchanged. This strategy combines DNA walker powered by catalyzed hairpin assembly (CHA) reaction with proportional fluorescence signal output methods, which can effectively reduce the risk of generating false-positive signals. The developed DNA walker demonstrated excellent analytical sensitivity and specificity, with a detection limit of 60 pM. The DNA walker was also capable of selectively detecting H5N1 DNA in clinical blood serum samples, which demonstrated its potential for use in various clinical applications.

Anti-Fouling Magnetic Beads Combined with Signal Amplification Strategies for Ultra-Sensitive and Selective Electrochemiluminescence Detection of MicroRNAs in Complex Biological Media.

Herein, an electrochemiluminescence (ECL) microRNA biosensor based on anti-fouling magnetic beads (MBs) and two signal amplification strategies was developed. The newly designed anti-fouling dendritic peptide was wrapped on the surfaces of MBs to make them resistant to nonspecific adsorption of biomolecules in complex biological samples so as to realize accurate and selective target recognition. One of the amplification strategies was achieved through nucleic acid cycle amplification based on the DNAzyme on the surfaces of MBs. Then, the output DNA generated by the nucleic acid cycle amplification program stimulated the hybrid chain reaction (HCR) process on the modified electrode surface to generate the other amplification of the ECL response. Titanium dioxide nanoneedles (TiO2 NNs), as a co-reaction accelerator of the Ru(bpy)2(cpaphen)2+ and tripropylamine (TPrA) system, were wrapped with the electrodeposited polyaniline (PANI) on the electrode surface to enhance the ECL intensity of Ru(bpy)2(cpaphen)2+. The conducting polymer PANI can not only immobilize the TiO2 NNs but also improve the conductivity of the modified electrodes. The biosensor exhibited ultra-high sensitivity and excellent selectivity toward the detection of miRNA 21, with a detection limit of 0.13 fM. More importantly, with the anti-fouling MBs as a unique separation tool, this ECL biosensor was capable of assaying targets in complex biological media such as serum and cell lysate.

Spherical nucleic acids-based cascade signal amplification for highly sensitive detection of exosomes

Exosomes are regarded as a promising biomarker in the diagnosis of disease due to their close relationship with the change of physiology and pathology. However, it is still a hard challenge to come up with a highly sensitive method for the exosomes detection. Herein, we propose a spherical nucleic acids (SNAs)-based cascade signal amplification strategy for the exosomes detection with high sensitivity. In this method, SNAs anchoring on exosomes membrane can be extended to form polyT sequence by terminal deoxynucleotidyl transferase (TdT), generating a template strand for the Exo III-catalyzed excision of the designed signal probe (probe A), which may finally induce significant decrease of electrochemical signal due to the consumption of probe A. Benefiting from the SNAs-based cascade signal amplification, this fabricated biosensor achieves a limit of detection for exosomes as low as 44 particles/μL. Moreover, this method shows good performance in the differentiation of healthy and malignancy colorectal cancer patients. Therefore, without complicated nucleic acids sequences design, our approach provides a cascade signal amplification strategy for the highly sensitive detection of exosomes and shows the potential applications in clinical diagnosis.

Ultrasensitive detection of trace Hg2+ by SERS aptasensor based on dual recycling amplification in water environment

Due to the nonbiodegradability and accumulation of mercury ion, even in extremely small amount, it will cause varying degrees of harm to environment and human health. Although researchers have developed many strategies to detect and monitor trace Hg2+, only a few provide sensitivities of less than 1.0 pM. Surface Enhanced Raman Spectroscopy (SERS) is a common method to detect mercury ion due to its high sensitivity, rapid detection and easy operation. In this work, we report a new SERS aptasensor based on dual recycling amplification for the detection of trace mercury ion, which combines SERS with nucleic acid signal amplification through functional aptamer and elaborately designed hairpin DNA. Under the optimal experimental conditions, this SERS aptasensor exhibits excellent selectivity and high sensitivity. A linear range (0.2–125 fM) and a low detection limit (0.11 fM) are obtained. By using specific aptamers, the strategy will provide a new idea for the trace detection of toxic contaminants in water environment.

A magnified aptamer fluorescence sensor based on the metal organic frameworks adsorbed DNA with enzyme catalysis amplification for ultra-sensitive determination of ATP and its logic gate operation

With the development of frame materials, metal organic frameworks (MOFs) have been successfully applied in the fields of biological small molecule analysis and fluorescent DNA detection. In this work, in view of the good adsorption characteristics of MIL-101(Cr), the highly sensitive detection of adenosine triphosphate (ATP) assisted nucleic acid exonuclease amplification by MIL-101(Cr) on the different affinity of single stranded DNA and double stranded DNA was investigated. The detection limit of ATP reaches 1.7 μM, and the platform has good applicability in biological samples. On this basis, an “AND” logic gate was successfully constructed. Superior sensitivity to ATP in the presence of exonuclease was reflected, which greatly enhanced the system’s fluorescence. Importantly, the fluorescence sensing application of this nanomaterial inspired other target detection and enriched the building blocks of fluorescence sensing platform.

Detection of the level of DNMT1 based on self-assembled probe signal amplification technique in plasma

DNA (cytosine-5)-methyltransferase1 (DNMT1) is the most abundant DNA methyltransferase in somatic cells, and it plays an important role in the initiation, occurrence, and rehabilitation of tumors. Herein, we developed a novel strategy for the detection of the level of DNMT1 in human plasma using the self-assembled nucleic acid probe signal amplification technology. In this method, the DNMT1 monoclonal antibody (McAbDNMT1) was immobilized on carboxyl magnetic beads to form immunomagnetic beads and then captured DNMT1 specifically. After that, DNMT1 polyclonal antibody (PcAbDNMT1) and biotinylated sheep anti-rabbit IgG (sheep anti rabbit IgG-Biotin) were sequentially added into the system to react with DNMT1 and form biotinylated double antibody sandwich immunomagnetic beads. In the presence of the bridging medium streptavidin, the biotinylated double antibody sandwich immunomagnetic beads would form a complex with biotinylated poly-fluorescein (Biotin-poly FAM), and the fluorescence intensity of the complex was proportional to the concentration of DNMT1. Immunomagnetic beads can capture the target DNMT1 in the sample, and Biotin-poly FAM can realize signal amplification. Using these strategies, we got a linear range of the system for DNMT1 level detection was from 2 nmol/L to 200 nmol/L, and the limit of detection (LOD) was 0.05 nmol/L. The method was successfully applied for the determination of DNMT1 in human plasma with the recovery of 101.3–106.0%. Therefore, this method has the potential for the detection of DNMT1 level in clinical diagnosis.

Detection of Treponema pallidum DNA in Oropharyngeal Swabs and Whole Blood for Syphilis Diagnosis.

Syphilis diagnosis relies on serological tests, which may be falsely nonreactive or may be reactive but not reflect current syphilis. Polymerase chain reaction for detection of T. pallidum DNA was performed on 123 oropharyngeal swabs, 120 whole bloods, and 46 lesion exudate swabs from 123 untreated individuals with syphilis (cases); oropharyngeal swabs from 148 at-risk controls without syphilis; and 73 oropharyngeal swabs and 36 whole bloods from 73 individuals recently treated for syphilis. Most (90.2%) cases had early syphilis. T. pallidum DNA was detected in 33 (26.8%) of 123 oropharyngeal swabs, 32 (26.7%) of 120 bloods, and 30 (65.2%) of 46 lesion exudate swabs. T. pallidum DNA was detected in 49 (40.8%) of 120 individuals in whom both oropharyngeal swabs and blood were tested. T. pallidum was more likely to be amplified from oropharyngeal swabs when it was amplified from blood than when it was not (15 of 32 [46.9%] vs. 17 of 88 [19.3%], P = 0.003). For each 2-fold increase in serum rapid plasma reagin titer, the odds of detection of T. pallidum DNA in oropharyngeal swabs increased by 1.44 (95% confidence interval, 1.14-1.82, P = 0.003). T. pallidum DNA was not detected in oropharyngeal samples from controls, but it was detected in 3 (8.3%) of 36 bloods from individuals recently treated for syphilis: 2 at 1 day and 1 at 5 days after initiation of syphilis treatment. Nucleic amplification tests can identify recent T. pallidum infection and may be particularly useful for diagnosis of very early or asymptomatic syphilis.

Comparison of the Efficacy of the Cartridge-Based Nucleic Amplification (CBNAAT)/Xpert Test and Histology of Genital Tissues in Diagnosing Female Genital Tuberculosis.

BackgroundDiagnosing female genital tuberculosis (FGTB) is very difficult by routine laboratory investigations. Collecting tissues from genital structures, especially from tubes for histology, is impossible. The cartridge-based nucleic amplification (CBNAAT)/Xpert RIF test is a new polymerase chain reaction (PCR)-based method that is quick and may diagnose FGTB from any tissue type; however, it should not be contaminated with blood. This study was conducted to compare the efficacy of CBNAAT and the histology of genital tissue in suspected cases.Materials and methodsThis was a prospective study of the diagnostic efficacy of 91 cases of suspected FGTB randomly selected from March 2018 to September 2019 at a rural tertiary care center. Endometrial tissue collected in 86 patients (59 infertility, 27 menstrual irregularities) and tubal/peritoneal tissue from hysterectomy or laparotomy specimens of five participants who underwent surgery were sent for histopathological analysis and CBNAAT and the results were evaluated and compared.ResultsThere were 59 (64.83%) and 32 (35.2%) cases of infertility and menstrual irregularities, respectively. Primary infertility (38; 41.75%) was the most common complaint. Endometrial biopsies (EB) of two (2.23%) cases were found positive for tuberculosis (TB) both on histopathological examination (HPE) and CBNAAT. In addition, both patients had primary infertility. Of the 32 cases with menstrual abnormalities (27 EB and three tubal tissue, two peritoneal and nodular tissue), none were found to be positive for TB on HPE or CBNAAT. A highly significant association was found between histopathology and CBNAAT (p<0.0001) in the endometrial tissue of infertile patients. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 100% for CBNAAT, with reference to histopathology.ConclusionWe recommend CBNAAT for the early detection of FGTB, with the added advantage of early results, minimal technical expertise, and detection of drug-resistant tuberculosis (TB).

Microbiota of a UASB Reactor Treating Palm Oil Mill Effluent Using HiSeq Sequencing

Potential microbial communities in the UASB reactor fed with two different substrates i.e synthetic wastewater and raw palm oil mill effluent (POME) were elucidated by using one of the most popular techniques in molecular methods, viz 16S rDNA cloning. The methodology involved; the extraction of nucleic acids, amplification and cloning of the 16S rRNA genes on sequencing HiSeq platform and finally identification and affiliation of the isolated clone with the aid of phylogenetic software. Results showed that the genus methanosarcina and methanosaeta were dominant methanogens in this study for both substrates types. Overall, microbial population (Bacteria and Archaea) in sample A (POME as substrate) is more diverse compared to sample B (synthetic wastewater as substrate) due to abundance of microorganism population in raw POME which was used as a substrate. However for the methanogenic (Archaea) diversity in both samples, there was not much different between sample A and sample B probably due to similar inoculum was inoculated in the reactor despite of have using different substrate type.

SARS-CoV-2 RT-PCR positivity and antibody prevalence among asymptomatic hospital-based health care workers.

BackgroundThe level of asymptomatic infection with SARS-CoV-2 could be substantial and among health care workers (HCWs) a source of continuing transmission of the virus to patients and co-workers.ObjectivesMeasure the period prevalence of SARS-CoV-2 PCR positivity and seroprevalence of SARS-CoV-2 IgG antibodies among a random sample of asymptomatic health system hospital-based health care workers (HCWs) 6½ -15½ weeks after 4/5/2020, the peak of the first surge of COVID-19 admissions.ResultsOf 524 eligible and consented participants from four metropolitan hospitals, nasopharyngeal swabs were obtained from 439 (83.8 %) and blood from 374 (71.4 %). Using PCR nucleic acid-based amplification (NAAT) methods, the period prevalence of SARS-CoV-2 infection was 0.23 % (95 % confidence interval (CI) 0.01 %–1.28 %; 1/439) from 5/21/20−7/16/20. The seroprevalence of SARS-CoV-2 IgG antibodies from June 17-July 24, 2020 was 2.41 % (95 % CI 1.27 %–4.51 %; 9/374). Those who were reactive were younger (median age 36 versus 44 years; p = 0.050), and those with self-reported Hispanic/Latino ethnicity had a higher seroprevalence (2/12 = 16.7 % versus 7/352 = 2.0 %; p = 0.051). There were no significant differences by sex, race, residence, hospital, unit or job type. The one employee who was found to be PCR test positive in this study was also reactive for IgG antibodies, tested 27 days later.ConclusionsThe period prevalence of PCR positivity to SARS-CoV-2 and IgG seroprevalence was unexpectedly low in asymptomatic HCWs after a peak in COVID-19 admissions and the establishment of state and institutional infection control policies, suggesting that routine screening tests while community prevalence is relatively low would produce a minimal yield.

Whole genome characterization of strains belonging to the Ralstonia solanacearum species complex and in silico analysis of TaqMan assays for detection in this heterogenous species complex

Identification and classification of members of the Ralstonia solanacearum species complex (RSSC) is challenging due to the heterogeneity of this complex. Whole genome sequence data of 225 strains were used to classify strains based on average nucleotide identity (ANI) and multilocus sequence analysis (MLSA). Based on the ANI score (>95%), 191 out of 192(99.5%) RSSC strains could be grouped into the three species R. solanacearum, R. pseudosolanacearum, and R. syzygii, and into the four phylotypes within the RSSC (I,II, III, and IV). R. solanacearum phylotype II could be split in two groups (IIA and IIB), from which IIB clustered in three subgroups (IIBa, IIBb and IIBc). This division by ANI was in accordance with MLSA. The IIB subgroups found by ANI and MLSA also differed in the number of SNPs in the primer and probe sites of various assays. An in-silico analysis of eight TaqMan and 11 conventional PCR assays was performed using the whole genome sequences. Based on this analysis several cases of potential false positives or false negatives can be expected upon the use of these assays for their intended target organisms. Two TaqMan assays and two PCR assays targeting the 16S rDNA sequence should be able to detect all phylotypes of the RSSC. We conclude that the increasing availability of whole genome sequences is not only useful for classification of strains, but also shows potential for selection and evaluation of clade specific nucleic acid-based amplification methods within the RSSC.

A sensitive platform for DNA detection based on organic electrochemical transistor and nucleic acid self-assembly signal amplification.

Highly sensitive detection of DNA is of great importance for the detection of genetic damage and errors for the diagnosis of many diseases. Traditional highly sensitive organic electrochemical transistor (OECT)-based methods mainly rely on good conductivity materials, which may be limited by complex synthesis and modification steps. In this work, DNA biosensor based on OECT and hybridization chain reaction (HCR) signal amplification was demonstrated for the first time. Au nanoparticles were electrochemically deposited on the Au gate electrode to increase the surface area. Then, the HCR products, long negatively charged double-stranded DNA, were connected to the target by hybridization, which can increase the effective gate voltage offset of OECT. This sensor exhibited high sensitivity and even 0.1 pM target DNA could be directly detected with a significant voltage shift. In addition, it could discriminate target DNA from the mismatched DNA with good selectivity. This proposed method based on HCR in DNA detection exhibited an efficient amplification performance on OECT, which provided new opportunities for highly sensitive and selective detection of DNA.

A simple and low-cost paper-based colorimetric method for detecting and distinguishing the GII.4 and GII.17 genotypes of norovirus

In modern times, viruses still threaten people's lives. Among them, norovirus was the main pathogenic factor in the cause of gastroenteritis and foodborne illness, of which the GII.4 and GII.17 genotypes are prevalent in China and most parts of the world. A simple and low-cost platform for rapid and accurate norovirus detection remains a major challenge. After the cell-free system and paper-based chromogenic system were optimized, a rapid and specific norovirus detection method was established based on norovirus-specific sequences in combination with toehold switch elements. The development of a visible color change during detection eliminates the need for any complicated instruments. We validated this strategy and its specificity in differentiating GII.4, GII.17, Zika virus, and human coronavirus HKU1. The results showed that the optimized detection system not only provided a simple and rapid detection method for the sufficient differentiation of the two norovirus genotypes but also showed high specificity and no cross-reactivity with other viruses. Using nucleic acid isothermal amplification, this assay showed a limit of detection of 0.5 pM for the GII.4 genotype and 2.6 fM for the GII.17 genotype in reactions that could be observed directly with the naked eye. Our results suggested that this paper-based colorimetric method could serve as a simple and low-cost visual detection method for pathogens in clinical samples, especially in remote or rural areas.

The pine wood nematode Bursaphelenchus xylophilus and molecular diagnostic methods

Bursaphelenchus xylophilus, commonly known as the pine wood nematode (PWN), is one of the most destructive nematode species. It causes pine wilt, a slow progressive disease that results in economic loss worldwide. Here, we review the nematode detection methods, including morphological, molecular, chemical, and protein-based methods, and assess how the nematode’s distribution, virulence, incidence, and severity may be attenuated. Pathogenicity of the pine wood nematode is determined via its morphological and molecular characteristics. Many studies have used nucleic acid PCR amplification as a molecular-based technique, but there are often problems with DNA isolation. Currently, molecular-, chemical-, and protein-based analysis methods are used to diagnose emerging pine wilt diseases. It is important to quickly diagnose and treat symptomatic trees, but the asymptomatic trees also require quick diagnosis and removal. For this reason, morphological- and DNA-based methods should be combined with chemical- and protein-based approaches for quick and efficient detection.

Application of Multiplexed Aptasensors in Food Contaminants Detection

The existence of contaminants in food poses a serious threat to human health. In recent years, aptamer sensors (aptasensors) have been developed rapidly for the detection of food contaminants because of their high specificity, design flexibility, and high efficiency. However, the development of high-throughput, highly sensitive, on-site, and cost-effective methods for simultaneous detection of food contaminants is still restricted due to multiple signal overlap or mutual interference and cross-reaction between different analytes with similar molecular structures. To overcome these problems, this Review summarizes some effective strategies from the articles published in recent years about multiplexed aptasensors for the simultaneous detection of food contaminants. This work focuses on the application of multiplexed aptasensors to simultaneously detect antibiotics, pathogens, and mycotoxins in food. These aptasensors mainly contain fluorescent aptasensors, electrochemical aptasensors, surface-enhanced Raman scattering-based aptasensors, microfluidic chip aptasensors, and paper-based multiplexed aptasensors. In addition, this Review also covers the application of nucleic acid cycle amplification and nanomaterial amplification strategies to improve the detection sensitivity. Finally, the limitations and challenges in the design of multiplexed aptasensor are also taken into account.

Specificity Enhancement of Deoxyribonucleic Acid Polymerization for Sensitive Nucleic Acid Detection.

Specificity of DNA polymerization plays a critical role in DNA replication and storage of genetic information. Likewise, biotechnological applications, such as nucleic acid detection, DNA amplification, and gene cloning, require high specificity in DNA synthesis catalyzed by DNA polymerases. However, errors in DNA polymerization (such as mis-incorporation and mis-priming) can significantly jeopardize the specificity. Herein, we report our discovery that the specificity of DNA enzymatic synthesis can be substantially enhanced (up to 100-fold higher) by attenuating DNA polymerase kinetics via the phosphorothioate dNTPs. This specificity enhancement allows convenient and sensitive nucleic acid detection, polymerization, PCR, and gene cloning with complex systems (such as human cDNA and genomic DNA). Further, we found that the specificity enhancement offered higher sensitivity (up to 50-fold better) for detecting nucleic acids, such as COVID-19 viral RNAs. Our findings have revealed a simple and convenient strategy for facilitating specificity and sensitivity of nucleic acid detection, amplification, and gene cloning.