Strand Exchange Amplification Research Articles

Comparison of four isothermal amplification techniques: LAMP, SEA, CPA, and RPA for the identification of chicken adulteration

In recent years, with growing concern worldwide about meat and meat product adulteration, dozens of DNA-based isothermal amplification techniques have been developed and applied to detect meat adulteration. Thus, we evaluated four mainstream isothermal amplification techniques, including loop-mediated isothermal amplification (LAMP), denaturation bubble-mediated strand exchange amplification (SEA), cross-priming amplification (CPA), and recombinase polymerase amplification (RPA) focusing on the limit of detection, simplicity, amplification time and cost to confirm the most effective and suitable method for point-of-care testing (POCT) of chicken and chicken product authenticity. The LAMP, CPA, and RPA primers all targeted the chicken mitochondrial cytochrome b gene. The SEA primers were provided by the SEA kit. All methods showed good specificity to chicken. Among them, LAMP, CPA, and RPA have the lowest detection limit, with 1.2 pg μL−1. As low as 0.1% chicken in mutton can be detected in LAMP and RPA methods. Although RPA costs 10 times more than LAMP, the system and primers of LAMP are too complex. Therefore, it must be admitted that RPA is the most suitable method in multiplex detection, and LAMP is much better than the other three methods in single-plex detection. Our research provides an excellent reference value for the rapid on-site detection of meat and meat product adulteration.

Boron nitride nanoplate-based strand exchange amplification with enhanced sensitivity and rapidity for quantitative detection of Staphylococcus aureus in food samples.

Staphylococcus aureus is one of the most common foodborne pathogens that can cause serious food poisoning and infectious diseases in humans. Standard identification approaches include nucleic acid amplification, but current amplification tools suffer from low amplification efficiency, resulting in the risk of low sensitivity and long detection time. Herein, boron nitride nanoplates (BNNPs) were chosen as an additive for enhancing the sensitivity and rapidity of strand exchange amplification (SEA), thereby successfully expanding the application of nucleic acid detection for detecting Staphylococcus aureus in food samples. As a result, SEA based on boron nitride nanoplates (BNNP-SEA) was employed for sensitive and rapid detection of foodborne pathogen Staphylococcus aureus. Compared with classical SEA, the BNNP-based SEA assay was more than 10-fold sensitive, and the detection time was reduced by 15 minutes. The optimized BNNP-based SEA shows a wide linear range from 40 pg to 50 ng in a diluted solution of the target DNA with a low detection limit of 40 pg. Moreover, the BNNP-based SEA achieves the quantitative detection of Staphylococcus aureus in different food samples (pork, beef, mutton, duck, milk and shrimp). In contrast to the classical SEA, the BNNP-based SEA method enabled sensitive and rapid detection of Staphylococcus aureus in the above food samples at concentrations as low as 5 × 103 CFU mL-1. The BNNP-based SEA assay is specific, sensitive and reliable, offering a valuable diagnostic technology for routine analysis in food safety research.

A biphasic accelerated strand exchange amplification strategy for culture-independent and rapid detection of Salmonella enterica in food samples

Salmonella enterica is a common foodborne pathogen that can cause food poisoning in humans. The organism also infects and causes disease in animals. Rapid and sensitive detection of S. enterica...

The detection of Bursaphelenchus xylophilus via accelerated strand exchange amplification: An ultra‐rapid and accurate method

AbstractOne of the most damaging pathogens of pinewood is the pinewood nematode, Bursaphelenchus xylophilus, which could cause an adverse effect on the ecosystems of forests and the commerce of timber. Therefore, it is crucial to realize rapid and accurate B. xylophilus detection. In this work, an accelerated strand exchange amplification method (ASEA) was established to detect B. xylophilus for the first time. By integrating with fast nucleic acid extraction, the whole detection procedure could be finished within 30 min, dramatically shortened the detection time. The ASEA method exhibited high specificity towards B. xylophilus and the detection limit for B. xylophilus plasmid DNA was as low as 1.0 × 100 copies/μL. Furthermore, the ASEA approach also exhibited accurate detection for B. xylophilus when applied to actual pinewood samples, meeting the demand of B. xylophilus detection in realistic scenario. We believe the ASEA method has significant potential for B. xylophilus detection, and it will be helpful for controlling forest pest and quarantine regulations.

Rapid quantitative detection of Klebsiella pneumoniae in infants with severe infection disease by point-of-care immunochromatographic technique based on nanofluorescent microspheres.

Background: Klebsiella pneumoniae (KP, K. pneumoniae) is one of the most important nosocomial pathogens that cause severe respiratory infections. As evolutionary high-toxic strains with drug resistance genes increase year by year, the infections caused by it are often accompanied by high mortality, which may be fatal to infants and can cause invasive infections in healthy adults. At present, the traditional clinical methods for detecting K. pneumoniae are cumbersome and time-consuming, and the accuracy and sensitivity are not high. In this study, nanofluorescent microsphere (nFM)-based immunochromatographic test strip (ICTS) quantitative testing platform were developed for point-of-care testing (POCT) method of K. pneumoniae. Methods: 19 clinical samples of infants were collected, the genus-specific gene of mdh was screened from K. pneumoniae. Polymerase chain reaction (PCR) combined with nFM-ICTS based on magnetic purification assay (PCR-ICTS) and strand exchange amplification (SEA) combined with nFM-ICTS based on magnetic purification assay (SEA-ICTS) were developed for the quantitative detection of K. pneumoniae. The sensitivity and specificity of SEA-ICTS and PCR-ICTS were demonstrated by the existing used classical microbiological methods, the real-time fluorescent quantitative PCR (RTFQ-PCR) and PCR assay based on agarose gel electrophoresis (PCR-GE). Results: Under optimum working conditions, the detection limits of PCR-GE, RTFQ-PCR, PCR-ICTS and SEA-ICTS are 7.7 × 10-3, 2.5 × 10-6, 7.7 × 10-6, 2.82 × 10-7ng/μL, respectively. The SEA-ICTS and PCR-ICTS assays can quickly identify K. pneumoniae, and could specifically distinguish K. pneumoniae samples from non-K. pneumoniae samples. Experiments have shown a diagnostic agreement of 100% between immunochromatographic test strip methods and the traditional clinical methods on the detection of clinical samples. During the purification process, the Silicon coated magnetic nanoparticles (Si-MNPs) were used to removed false positive results effectively from the products, which showed of great screening ability. The SEA-ICTS method was developed based on PCR-ICTS, which is a more rapid (20min), low-costed method compared with PCR-ICTS assay for the detection of K. pneumoniae in infants. Only need a cheap thermostatic water bath and takes a short detection time, this new method can potentially serve as an efficient point-of-care testing method for on-site detection of pathogens and disease outbreaks without fluorescent polymerase chain reaction instruments and professional technicians operation.

A visual denaturation bubble-mediated strand exchange amplification and RGB visual analysis-based assay for quantitative detection of Helicobacter pylori in saliva.

Helicobacter pylori (H. pylori) is a class I carcinogen causing gastric cancer. Almost 50% of people on earth have been infected and it is worse in developing countries. Early diagnosis of H. pylori infection is the most important strategy for preventing the spread and worse consequences. H. pylori can be isolated from human saliva, and the sampling of saliva is easy and convenient. Therefore, we developed a visual denaturation bubble-mediated strand exchange amplification and RGB visual analysis-based assay for quantitative detection of H. pylori in saliva in this study. Under the optimized reaction temperature and time, the SEA reaction could be finished in 30min with a simple reaction system and low dependency on equipment. The detection results could be qualitatively identified by the naked eye and quantitatively analyzed by a developed RGB visual analysis method. The limit of detection (LOD) of RGB visual analysis was 10.8CFU/mL. This assay had good specificity and anti-interference capacity. In the artificial contamination test, the recovery rate of our assay was between 99.3% and 111.5%, with RSD values ranging from 1.7% to 3.5%. These indicated our assay also had good reliability in the detection of saliva. We believe this assay showed good potential for better non-invasive diagnosis of H. pylori infection.

Denaturation bubble-mediated two-stage isothermal nucleic acid amplification in a single closed tube.

Here, we established a two-stage isothermal amplification method comprising strand exchange amplification and hyperbranched rolling circle amplification, which only employed one DNA polymerase and was performed in a single closed tube at a constant temperature, providing a promising signal amplification strategy for accurate and rapid pathogen detection in the clinic.

Rapid, specific and sensitive detection of Vibrio parahaemolyticus in seafood by accelerated strand exchange amplification.

Vibrio parahaemolyticus infectious diseases caused by seafood contamination may be life-threatening to people with weak immunity. The detection of the Vibrio parahaemolyticus pathogen in aquatic foods is critical for reducing the outbreak of human Vibrio parahaemolyticus-associated diseases. In this study, a highly sensitive, specific, and time-saving real-time narrow thermal-cycling amplification detection method was developed based on accelerated strand exchange amplification (ASEA). It can detect cultured Vibrio parahaemolyticus at concentrations as low as 25 CFU mL-1. In addition, for artificially spiked scallop meat, the detection limit was 1.8 × 103 CFU g-1 without pre-culture and 18 CFU g-1 of initial inoculum after 3 h enrichment. The whole assay, starting from DNA extraction, can be completed within 20 min. The ASEA detection method established in this study is an effective tool for the rapid detection of Vibrio parahaemolyticus strains in a large number of seafood samples.

Rapid diagnosis of acute myocardial infarction based on reverse transcription-accelerated strand exchange amplification of miR-208a.

Acute myocardial infarction (AMI) is a prevalent cardiovascular disease associated with high morbidity and mortality, posing a significant threat to human health. Therefore, early diagnosis of AMI has become a focal point of research. MiR-208 is specifically expressed in the heart and is involved in the regulation of cardiomyocyte hypertrophy, cardiac fibrosis, and other myocardial gene expressions. It is expected to be applied in the clinical detection of AMI due to its release by damaged myocardial cells within 3 hours of AMI. In this study, we developed a denatured bubble-mediated reverse transcription-accelerated strand exchange amplification (RT-ASEA) method to detect the early biomarker miR-208a of AMI. The novel approach allowed rapid amplification of miR-208a in 15 minutes, with good performance in terms of repeatability (CV < 6%), determination limit (1 × 100 pmol L-1), and linearity (R2 = 0.9690). Based on the analysis of 42 clinical samples, a strong correlation was observed between the Ct value of miR-208a detected by the RT-ASEA method and the cTnI concentration, considered the gold standard for diagnosis of AMI. The research suggested that the RT-ASEA method could be applied to distinguish between AMI and healthy groups. The area under the receiver operating characteristic curve (AUC) was 0.9976, with a sensitivity of 96% and a specificity of 100%. Optimized RT-ASEA is a reliable and efficient method for miRNA detection. Furthermore, this study provides crucial data support for the development of miR-208a as an early biomarker for AMI, which is of great significance in life and health.

Rapid and sensitive detection of Staphylococcus aureus via an all-in-one staggered strand exchange amplification platform.

Staphylococcus aureus is a common foodborne pathogen that causes food poisoning and infectious diseases in humans and animals. Rapid detection of S. aureus with high sensitivity is of great significance to prevent the spread of this pathogen. In this study, we developed a staggered strand exchange amplification (SSEA) method by refining denaturation bubble-mediated strand exchange amplification (SEA) to detect S. aureus at a constant temperature with high specificity and efficiency. This method employs a DNA polymerase and two sets of forward and reverse primers arranged in tandem that invade denaturation bubbles of double-stranded DNA. In comparison, the sensitivity of SSEA was 20 times that of SEA. Subsequently, magnetic bead (MB)-based DNA extraction was introduced into SSEA to establish an all-in-one SSEA platform that incorporated sample processing, amplification and detection in a single tube. The use of MBs further enhanced the sensitivity of SSEA by two orders of magnitude. Specificity tests showed that the all-in-one SSEA could specifically identify S. aureus and had no cross-reaction with other common foodborne pathogens. For artificially spiked meat samples, the method could detect 1.0 × 102 CFU g-1S. aureus in pork and 1.0 × 103 CFU g-1 in either duck or scallop samples without a bacterial enrichment step. The entire assay can be completed sample-to-answer within 1 h. Thus, we believe that this easy-to-operate diagnostic platform enables sensitive and accurate detection of S. aureus and holds great promise for the food safety industry.

Amino-modified silica membrane capable of DNA extraction and enrichment for facilitated isothermal amplification detection of Mycoplasma pneumoniae

Herein, we developed a facile integrated Mycoplasma pneumoniae diagnosis platform by combining amino-modified silica membrane (AMSM)-based nucleic acids fast extraction and enrichment with colorimetric isothermal amplification detection. AMSM demonstrates a strong ability to capture and enrich nucleic acids in complicated biological matrices, and the purified AMSM/nucleic acids composite could be directly used to perform isothermal amplification including denaturation bubble-mediated strand exchange amplification (SEA) and loop-mediated isothermal amplification (LAMP) reactions. Through comparing clinical specimens, excellent performance of AMSM-based SEA assay with 93.33% sensitivity and 100% specificity relative to real-time PCR was observed, and for AMSM-based LAMP was 96.67% and 100%, respectively. The diagnostic procedure could be completed within 55 min, and the colorimetric-based visual result further alleviates the use of sophisticated equipment. The proposed approach possesses great potential as a simple and time-saving alternative for point-of-care testing (POCT) of M. pneumoniae in resource-limited regions.

Graphene oxide-assisted optimized narrow-thermal-cycling amplification for accurate detection of Salmonella spp.

Salmonella is a rod-shaped, Gram-negative zoonotic pathogen that poses a serious global socioeconomic and public health threat. Rapid and accurate detection of Salmonella spp. is critical for effective control of its infection. In this study, an accurate, sensitive and specific graphene oxide-assisted accelerated strand exchange amplification (GO-ASEA) method for rapid detection of Salmonella spp. was developed and validated. The detection limit of the GO-ASEA method was 8.6 × 101fg μL-1 of Salmonella genomic DNA or 1 × 101CFUg-1 of Salmonella in spiked chicken faeces free of pre-enrichment. And the GO-ASEA method could specifically detect Salmonella spp. without cross-reactivity with other enteric pathogens. In addition, the novel method achieved Salmonella detection within 30min and was validated using 209 clinical samples, showing its good clinical applicability. Therefore, the GO-ASEA method is a new optional tool for the rapid detection of pathogenic microorganisms, which is ideal for food safety monitoring and high-throughput detection.

An all-in-one nucleic acid enrichment and isothermal amplification platform for rapid detection of Listeria monocytogenes

Listeria monocytogenes is an infectious foodborne pathogen that greatly threatens the public health worldwide. A simple and sensitive detection of L. monocytogenes is extremely important in food safety industry. In this study, we developed an all-in-one platform, which consists of nucleic acid extraction, amplification and visual detection in a single tube. Following a simple bacterial lysis, the released DNA and RNA were highly enriched by a chitosan modified silica membrane (CMSM). Enriched nucleic acids were then directly used for isothermal strand exchange amplification (SEA) and colorimetric detection. The analytical feasibility of this platform was successfully determined by identifying L. monocytogenes with a specific set of primers, and experiments confirmed a detection limit of 1.0 × 102 CFU/mL in pure culture, while the limit of detection in L. monocytogenes spiked pork was 1.0 × 103 CFU/g without bacterial enrichment and 1.0 × 10° CFU/g following 4 h enrichment. The whole SEA assay takes an hour to complete, allows one-step DNA and RNA amplification and substantially improves the assay sensitivity. In addition, the colorimetric-based visual result readout developed in this study fully alleviates the use of sophisticated equipment. Therefore, the proposed method has great potential for rapid and on-site identification of L. monocytogenes in resource-restricted settings.

Signal-enhanced visual strand exchange amplification detection of African swine fever virus by the introduction of multimeric G-quadruplex/hemin DNAzyme.

African swine fever virus (ASFV) causes hemorrhagic infectious disease in pigs with a fatality rate of nearly 100%. In this study, we developed a visual strand exchange amplification detection assay for ASFV. In the presence of ASFV, DNA amplification products containing multimeric G-quadruplex sequences were amplified by strand exchange amplification. These G-quadruplexes, assembled with hemin to form DNAzyme, displayed enhanced significant "turned-on" colorimetric signals to indicate detection results. The results showed that dimeric DNAzyme had the best visualization effect. Under the optimal reaction parameters, there was a linear relationship between the absorbance of the reaction solution at 417nm and the logarithm of ASFV concentration ranged from 1 × 101 to 1 × 103 copies/μL, and the detection limit was 2.7 copies/μL. We hoped this visual assay could be helpful in the rapid and sensitive detection of ASFV, and the results of multimeric G-quadruplex/hemin DNAzyme could be helpful for the development of better visual detection assays.

Accurate, rapid and highly sensitive detection of African swine fever virus via graphene oxide-based accelerated strand exchange amplification

An accurate, rapid and highly sensitive graphene oxide-based accelerated strand exchange amplification method was developed for quantitative detection of African swine fever virus (ASFV).

An ultra-fast, one-step RNA amplification method for the detection of Salmonella in seafood.

Salmonella is one of the most common pathogens associated with food-borne illness resulting from seafood consumption. Herein, an accelerated strand exchange amplification (ASEA) requiring only a pair of primers and one polymerase was first reported for ultra-fast, one-step RNA amplification detection of Salmonella in seafood. The ASEA method could detect Salmonella typhimurium DNA in dilutions as low as 10 copies per reaction and displayed good specificity for Salmonella under the interference of a variety of food-borne pathogens. In particular, ASEA could detect RNA in one step without additional reverse transcription. The detection limit for Salmonella in artificially contaminated oyster was 1 CFU mL-1 following 12 h of enrichment. Moreover, excellent performance of this assay was observed with 99.02% consistency relative to real-time PCR through actual sample detection. Combined with the rapid nucleic acid extraction method, the entire detection process could be completed within 20 min. Therefore, this assay opens up new prospects for the detection of food-borne pathogens in seafood with its rapidity, which would be very beneficial for food safety supervision and pathogen detection of high-throughput samples.

Nucleic acid extraction without electrical equipment via magnetic nanoparticles in Pasteur pipettes for pathogen detection

The outbreak of COVID-19 makes epidemic prevention and control become a growing global concern. Nucleic acid amplification testing (NAAT) can realize early and rapid detection of targets, thus it is considered as an ideal approach for detecting pathogens of severe acute infectious diseases. Rapid acquisition of high-quality target nucleic acid is the prerequisite to ensure the efficiency and accuracy of NAAT. Herein, we proposed a simple system in which magnetic nanoparticles (MNPs) based nucleic acid extraction was carried out in a plastic Pasteur pipette. Different from traditional approaches, this proposed system could be finished in 15 min without the supports of any electrical instruments. Furthermore, this system was superior to traditional MNPs based extraction methods in the aspects of rapid extraction and enhancing the sensitivity of a NAAT method, accelerated denaturation bubbles mediated strand exchange amplification (ASEA), to the pathogens from various artificial samples. Finally, this Pasteur pipette system was utilized for pathogen detection in actual samples of throat swabs, cervical swabs and gastric mucosa, the diagnosis results of which were identical with that provided by hospital. This rapid, easy-performing and efficiency extraction method ensures the applications of the NAAT in pathogen detection in regions with restricted resources.

Minimizing Leakage in Stacked Strand Exchange Amplification Circuits.

Signal amplification is ubiquitous in biology and engineering. Protein enzymes, such as DNA polymerases, can routinely achieve >106-fold signal increase, making them powerful tools for signal enhancement. Considerable signal amplification can also be achieved using nonenzymatic, cascaded nucleic acid strand exchange reactions. However, the practical application of such kinetically trapped circuits has so far proven difficult due to uncatalyzed leakage of the cascade. We now demonstrate that strategically positioned mismatches between circuit components can reduce unprogrammed hybridization reactions and therefore greatly diminish leakage. In consequence, we were able to synthesize a three-layer catalytic hairpin assembly cascade that could operate in a single tube and that yielded 3.7 × 104-fold signal amplification in only 4 h, a greatly improved performance relative to previous cascades. This advance should facilitate the implementation of nonenzymatic signal amplification in molecular diagnostics, as well as inform the design of a wide variety of increasingly intricate nucleic acid computation circuits.

Rapid and easy quantitative identification of Cronobacter spp. in infant formula milk powder by isothermal strand-exchange-amplification based molecular capturing lateral flow strip

Food-borne pathogen detection is of great significance for guaranteeing food safety and human health. Herein, we have developed a new detection method for rapid and easy quantitative identification of Cronobacter spp. in infant formula milk powder. This method well couples the advantages of isothermal strand exchange amplification (SEA) and molecular capturing lateral flow strip (LFS). The presence of Cronobacter spp. DNA template can be isothermally and exponentially amplified by SEA with a pair of labeled functional primers without thermal controller device. The high amplification efficiency of SEA enables the accumulation of plentiful dual-labeled amplicons that can be captured by a molecular capturing LFS with the using of fluorescent microsphere (FM) as tagger, which can be excited at 365 nm and the signal at 625 nm can be acquired for the quantitative analysis. The Cronobacter spp. in the concentration from 101 to 106 cfu/mL can be accurately determined with the visual detection limit of 10 cfu/mL within less than 1 h even in the complex infant formula milk powder. We expect this unique SEA-LFS method with its favorable assay performance can be a powerful analytical protocol in the field of foodborne pathogen detection.

Tri-primer-enhanced strand exchange amplification combined with rapid lateral flow fluorescence immunoassay to detect SARS-CoV-2.

The novel coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world, which has exposed humanity to unprecedented economic, social and health impacts. To achieve efficient and accurate detection of SARS-CoV-2 on site, we developed and verified a rapid and sensitive fluorescence lateral flow immunoassay based on the innovative enhanced strand exchange amplification (ESEA-LFIA) in this study. With good amplification efficiency for short-sequence targets, ESEA is an ideal choice for the point-of-care testing of SARS-CoV-2 with a high mutation rate. ESEA reaction can be completed in one step and verified by restriction enzyme digestion. The design consisting of three working primers greatly improved the amplification efficiency. Amplification of the target sequences of the RdRP and N genes can be accomplished under the same reaction conditions, and does not require expensive instruments. The sensitivity of the ESEA-LFIA assay targeting the RdRP and N genes was 90 copies per μL and 70 copies per μL, respectively. Specificity tests showed that the novel assay can specifically detect SARS-CoV-2, and had no cross-reactivity with 9 closely-related human pathogenic coronaviruses and other common respiratory pathogens with similar clinical manifestations. The cutoff values of the RdRP and N gene assays are 11 and 12, respectively, and the assays can be completed within 1 h. The novel strategy proposed in this study is a sensitive and specific method for the rapid detection of SARS-CoV-2, and is suitable as an effective potential bioanalytical tool to respond to future regional or global outbreaks of emerging infectious pathogens with high mutation rates.