Strand Exchange Amplification Research Articles

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.

Detection of Epstein-Barr virus by a rapid and simple accelerated denaturation bubble-mediated strand exchange amplification method.

Epstein-Barr Virus (EBV) is a tumor-associated virus naturally transmitted through saliva. This virus is the pathogen of infectious mononucleosis, which is closely related to the occurrence of nasopharyngeal carcinoma (NPC) and childhood lymphoma. Although a majority of EBV infected individuals exhibited good tolerance after primary infection, those who carry a viral load greater than the clinical cutoff value (COV), the upper level in healthy carriers, still suffer a high risk of cancer. Herein, a simple, rapid, and effective method, accelerated strand exchange amplification (ASEA), was developed for EBV detection, which could offer a strategy for non-invasive testing of EBV in saliva samples instead of blood samples as in traditional serology based methods and avoid bleeding during diagnosis. This approach could distinguish the genomic DNA of EBV and other species in saliva, and its limit of detection was as low as 1000 copies per mL, which was lower than the COV of EBV. Moreover, DNA extracted from saliva samples (n = 50) was employed as a template for EBV detection via qPCR and ASEA, the result of which showed that ASEA exhibited comparable sensitivity and specificity for actual sample diagnosis. Additionally, similar to conventional PCR, this method requires only one pair of primers and could be performed using a conventional fluorescence instrument, which makes this method easy to accomplish. Therefore, this rapid and effective method has the potential to provide rapid screening platforms for individuals with a high EBV load.

Accurate, rapid and low-cost diagnosis of Mycoplasma pneumoniae via fast narrow-thermal-cycling denaturation bubble-mediated strand exchange amplification.

Mycoplasma pneumoniae is a strong infectious pathogen that may cause severe respiratory infections. Since this pathogen may possess a latent period after infection, which sometimes leads to misdiagnosis by traditional diagnosis methods, the establishment of a rapid and sensitive diagnostic method is crucial for transmission prevention and timely treatment. Herein, a novel detection method was established for M. pneumoniae detection. The method, which improves upon a denaturation bubble-mediated strand exchange amplification (SEA) that we developed in 2016, is called accelerated SEA (ASEA). The established ASEA achieved detection of 1% M. pneumoniae genomic DNA in a DNA mixture from multiple pathogens, and the limit of detection (LOD) of ASEA was as low as 1.0 × 10−17 M (approximately 6.0 × 103 copies/mL). Considering that the threshold of an asymptomatic carriage is normally recommended as 1.0 × 104 copies/mL, this method was able to satisfy the requirement for practical diagnosis of M. pneumoniae. Moreover, the detection process was finished within 20.4 min, significantly shorter than real-time PCR and SEA. Furthermore, ASEA exhibited excellent performance in clinical specimen analysis, with sensitivity and specificity of 96.2% and 100%, respectively, compared with the “gold standard” real-time PCR. More importantly, similar to real-time PCR, ASEA requires only one pair of primers and ordinary commercial polymerase, and can be carried out using a conventional fluorescence real-time PCR instrument, which makes this method low-cost and easy to accomplish. Therefore, ASEA has the potential for wide use in the rapid detection of M. pneumoniae or other pathogens in large numbers of specimens. Graphical abstract

Detection of canine parvovirus and feline panleukopenia virus in fecal samples by strand exchange amplification.

Canine parvovirus 2 (CPV-2) and feline panleukopenia virus (FPLV) often cause acute enteric disease in their hosts. A simple, rapid, and effective method for the on-site detection of these viruses would be useful. We used a denaturation bubble-mediated strand exchange amplification (SEA) method to successfully detect CPV-2 and FPLV in fecal samples. SEA could detect as little as 3.6 pg/μL of CPV-2 and 6.6 pg/μL of FPLV genomic DNA following a 40-min incubation at an isothermal temperature of 61°C. Unlike PCR, SEA does not require complicated equipment, and positive samples produce a color change that can be visualized by the naked eye. Additionally, SEA is simpler than PCR because no extraction is needed, and heating of the fecal sample at 98°C can be performed with a heating block or water bath. This rapid and effective nucleic acid detection platform could be used as a point-of-care test for the detection of CPV-2 and FPLV.

A visual on‐site method for African swine fever virus detection in raw pig tissues

AbstractThe spread of African swine fever virus (ASFV) leads to high mortality in domestic pigs, and has caused huge losses in the global pork industry. Therefore, the rapid detection of ASFV is essential for the prevention and control of the epidemic. Herein, a denaturation bubble‐mediated strand exchange amplification (SEA) with colorimetric detection strategy for the rapid detection of ASFV was established and the results were visible with the naked eyes. The entire detection process based on isothermal amplification was performed on a portable metal bath only. And the whole process including rapid extraction and visual detection was finished within 50 min. The limit of detection of this method reached to 104 copies/μl of plasmid containing p72 gene. And the ASFV was successfully detected in eight different tissue and seven types of pig feed which revealed a strong anti‐interference ability in the presence of complex matrix. Through comparing 94 field positive and negative ASFV samples, excellent performance of SEA assay with 92.86% sensitivity and 100% specificity relative to PCR was observed. The developed method could be used for on‐site detection of ASFV with simplicity, rapidity, and visuality, especially suitable for pork industry food safety and ASFV control in resource‐limited areas.

Integrated silica membrane-based nucleic acid purification, amplification, and visualization platform for low-cost, rapid detection of foodborne pathogens.

Real-time fluorescence detection of nucleic acid exhibit excellent performance in analytical and diagnostic applications. However, the requirement of laboratory-based instrument and complex nucleic acid extraction greatly limits their application in point-of-care testing (POCT). Herein, a novel integrated silica membrane-based platform incorporating nucleic acid purification, amplification, and detection steps was developed. A universal and portable visualization platform was fabricated by incorporating denaturation bubble-mediated strand exchange amplification (SEA) reaction with silica membrane. The fluorescence signal of SYBR Green I with amplification products was visualized by the naked eye using a simple ultraviolet light on the silica membrane, and significant discrimination between the positive and negative samples could be easily and visually obtained. Besides, chitooligosaccharide-modified silica membrane allows the purification of nucleic acid in a totally aqueous system and enables in situ SEA. With the proposed integrated platform, 102-108cfu/mL Vibrio parahaemolyticus could be successfully detected and excellent performance was also revealed for gram-positive pathogens. The detection limit of the method for artificially spiked oysters was 103cfu/g and reached 100cfu/g after 12h enrichment. This proof-of-concept method could also be applied to a variety of nucleic acid amplification methods. We believe that the proposed silica membrane-based platform has great potential for the rapid and low-cost detection of nucleic acids especially in low-resource settings. Graphical abstract.

A novel isothermal detection method for the universal element of genetically modified soybean

Due to the increasingly heated debate on the potential threat of genetically modified (GM) crops to human health and environment, regulations and laws relevant to GM crops have been issued in many countries and regions to strictly restrict their cultivation and application. Therefore, fast and accurate method to realized on-site detection of GM crops is greatly demanded. In this work, a novel isothermal amplification method termed denaturation bubble-mediated strand exchange amplification (SEA) was proposed first time to detect GM crops. Fluorescence assay based on SEA could accurately distinguished GM and non-GM soybean by detecting agrobacterium tumefaciens nopaline synthase (NOS) terminator, which was widely incorporated in GM crops. Moreover, this feasible and specific method could detect NOS terminator from as low as 200 pg/μL total genomic DNA of GM soybean. In addition, in the actual sample detection, the result of colorimetric assay based on SEA results could be directly observed by the naked eyes within 58 min. Compared with the traditional methods based on PCR, which normally required complex equipment, skilled technicians and long operation time, this simple, fast and precise method is more desirable for the on-site GM crops detection.

A fully integrated hand-powered centrifugal microfluidic platform for ultra-simple and non-instrumental nucleic acid detection

Hand-powered centrifugal microfluidics combined with isothermal nucleic acid amplification testing (NAAT) have been one of the most promising rapid detection platforms in resource-limited settings. However, current hand-powered centrifuges still suffer from customized instrument-based operation and low rotation rate; and most isothermal NAAT were conducted with complicated reaction systems for DNA detection and required an additional step for RNA detection. Herein, we built a fully hand-powered centrifugal miniaturized NAAT platform inspired by buzzer toys, which embedded sample preparation, strand exchange amplification (SEA) and visual fluorescence detection together. The centrifugal disc was easily fabricated, and operated the mixing in 1 min by simply dragging the looped rope through it with a mean input force of 16.5 N, enabling its rotation rate reach 5000 rpm. In addition, SEA was an ultra-simple one-step DNA or RNA detection method initiated by Bst DNA polymerase and a pair of primers, and thus we took all its merits and integrate it into microfluidic systems firstly. Furthermore, taking Vibrio parahemolyticus as an example, the microfluidic platform achieved DNA or RNA detection within 1 h; and the detection limit of the microchip for artificially spiked oysters was 103 CFU/g without cumbersome sample preparation, and reached to 100 CFU/g after enrichment. Therefore, we provided an ultra-simple and non-instrumental microfluidic platform powered merely by hands, performing general potential in sample-to-answer NAAT for versatile pathogens in remote regions.

Highly sensitive visual detection of nucleic acid based on a universal strand exchange amplification coupled with lateral flow assay strip

Amplification reactions coupled with electrophoresis or real-time fluorescence system have been successfully used for the analysis of nucleic acid. However, complex procedures or expensive instruments greatly limit their application in low-resource settings. To address these shortcomings, we fabricated a universal and simple detection platform by integrating strand exchange amplification (SEA) with lateral flow assay (LFA) strip. SEA is a simple isothermal amplification reaction, only requires a pair of primers and one DNA polymerase, above all, its short amplicons are easy to migrate on the strip. LFA strip was proved to be stable for months without large signal deviations and result could be easy to read by naked eyes, which makes it an appropriate option for field-based analysis. Our proposed SEA-LFA strip could reliably detect as few as 0.05 nM pork DNA and 0.07 nM duck DNA by the naked eye, its analytical performance is comparable with laboratory-based real-time fluorescence test. Furthermore, this proof-of-concept method could be applied to detect a wide variety of nucleic acid by focusing on primer design rather than on the development of a wholly new analysis platform. We believe that this simple visualization system has great potential as a preliminary test tool in resource-limited areas.

Development of a direct and visual isothermal method for meat adulteration detection in low resource settings

Simple, fast and efficient method for on-site detection of meat adulteration is of increasing demand in low-resource regions. Here, a direct and visual denaturation bubble-mediated strand exchange amplification method (SEA) was developed to detect meat adulteration by targeting the mitochondrial sequence from duck, which required only a heating block. The method allowed detecting as low as 10 pg/μL duck DNA and 0.1% duck meat in binary mixtures, sufficiently meeting the demand of meat adulteration detection. Notably, the method realized instrument-free readout by significant color discrimination of positive and negative results. By coupling with fast DNA release, a direct SEA method was developed and reduced the whole detection time to 1 h, eliminating complex DNA extraction process. The developed method provided a promising strategy with integration of fast sample lysis, miniaturized reaction platform and visual readout for meat adulteration, especially suitable for low resource point-of-care settings.

Primer design strategy for denaturation bubble-mediated strand exchange amplification

Denaturation bubble-mediated strand exchange amplification (SEA) is a novel, rapid isothermal nucleic acid amplification has been applied for point-of-care molecular diagnostic in food safety, meat adulteration, forest disease and animal disease. Nevertheless, the absence of specialized strategy for SEA primers design led to long-time of primer screening progress before SEA reaction execution, which would largely increase the time consuming when SEA is utilized for detecting other new targets. In this present work, we investigated the impact of the following primers' attributes on SEA efficiency, including Tm value, 3′ end G/C content, self-complementary and 3′ complementary, according to which we demonstrated that optimal Tm value and reaction temperature were all 61 °C, while 3′-terminal nucleotide should be G/C, as the SEA reaction induced by the primers possessing these attributes exhibited significantly lower threshold time (Tt) value. Moreover, self-complementary and 3′ complementary of primers should be avoided. Besides, we also discussed the consideration priority order of these factors, which was self-complementary and 3′ complementary, Tm value and 3′ end G/C content in turn. Because the SEA primer design strategy is first presented, our work will greatly promote the application of SEA in point-of-care test.

Accelerated denaturation bubble-mediated strand exchange amplification for rapid and accurate detection of canine parvovirus.

Canine parvovirus (CPV), a strong infectious canine pathogen, has been recognized as a threat to canine health worldwide since the 1970s. Although convenient detection methods have been developed, such as the colloidal gold test strip, most of these methods are based on antibody detection, which is relatively ineffective for detecting pathogens during the incubation period. For institutions and businesses with many dogs, e.g., dog training centers and kennels, more sensitive detection methods are required to prevent the swift spread of CPV. Thus, we developed accelerated denaturation bubble-mediated strand exchange amplification (ASEA) for CPV detection, and it is a rapid, convenient, and cost-effective method. ASEA was able to distinguish CPV genomic DNA in a mixture that included canine genomic DNA as well as nucleic acids sourced from nine other common pathogens, with detection of target DNA as low as 8.0 × 10-18 M within 16.6 min. Coupled with the thermal lysis method modified by us that only requires 3 min to perform, the entire detection procedure can be completed within approximately 20 min and only requires a simple heating block and an ordinary fluorescence PCR instrument. Moreover, ASEA exhibited greater sensitivity than colloidal gold test strips in actual specimen detection. This technique is rapid, easy to perform, and highly sensitive, and therefore, this approach has the potential to rapidly detect CPV in institutions with large populations of dogs.

Lab in a Pasteur pipette: low-cost, rapid and visual detection of Bacillus cereu using denaturation bubble-mediated strand exchange amplification

Driven by a bright prospect for rapid, portable and cost-effective point-of-care testing, an assembled Pasteur pipette device to integrate nucleic acid extraction, amplification and detection was developed to detect B. cereus in a sample-to-answer format. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was chosen to perform isothermal amplification because it requires only a pair of primers and one Bst DNA polymerase. The established SEA can detect as low as 1.0 × 10−13 M genomic DNA of B. cereus, which was comparable with the previously reported method for B. cereus detection. The assembled Pasteur pipette allows sample-to-answer diagnostic in a simple, low-cost, portable, and disposable format. The inherent function of Pasteur pipette enables direct liquid handling without the need of extra pipettes, syringes or pumps. Visual readout was achieved by using a pH sensitive dye, further simplifying result judgment process. The detection limit for B. cereus is 1.0 × 104 CFU/mL in pure cultures, while the detection limit in artificially contaminated milk is 1.0 × 105 CFU/mL without enrichment and 1.0 × 100 CFU/mL following 12 h enrichment. Considering that typical cell counts in food samples associated to food poisoning are 1.0 × 105 to 1.0 × 108 CFU per gram/milliliter B. cereus, our Pasteur pipette is enough sensitive for answer-to-sample diagnosis of B. cereus even directly from foods without enrichment. The whole diagnostic procedure could be completed within 50 min, dramatically decreasing the detection time. In a word, the assembled Pasteur pipette device, combined with a homemade metal bath, possesses great potential for sample-to-answer application in resource-limited settings.

Rapid and Simple Detection of Viable Foodborne Pathogen Staphylococcus aureus.

Staphylococcus aureus (S. aureus) contamination in food safety has become a worldwide health problem. In this work, we utilized RNA one-step detection of denaturation bubble-mediated Strand Exchange Amplification (SEA) method to realize the detection of viable foodborne pathogen S. aureus. A pair of S. aureus specific primers were designed for the SEA reaction by targeting hypervariable V2 region of 16S rDNA and the amplification reaction was finished about 1 h. The results of amplification reaction could be observed by the naked eyes with a significant color change from light yellow to red to realize the colorimetric detection of S. aureus. Therefore, there only required an isothermal water bath, which was very popular for areas with limited resources. In real sample testing, although the SEA detection was so time-saving compared with the traditional plating method, the SEA method showed great consistency with the traditional plating method. In view of the above-described advantages, we provided a simple, rapid and equipment-free detection method, which had a great potential on ponit-of-care testing (POCT) application. Our method reported here will also provide a POCT detection platform for other food-borne pathogens in food, even pathogenic bacteria from other fields.

Rapid diagnosis of Mycoplasma pneumonia infection by denaturation bubble-mediated strand exchange amplification: comparison with LAMP and real-time PCR

M. pneumoniae infection is often ignored due to its similar clinical symptom with respiratory tract infections caused by bacteria or viruses, and thus leading to misdiagnosis and delayed treatment. It is critical to develop a rapid, sensitive and specific diagnosis method. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was established, which is an isothermal method with only a primer pair and one Bst DNA polymerase. Notably, colorimetric SEA assay was developed with simple visual readout, making instrument-independent in detection step. The method could detect as low as 1.0 × 104 copies/mL genomic DNA within 60 min. Considering that more than 80% infected patients have 1.0 × 105−1.0 × 107 copies/mL M. pneumonia DNA, SEA is available for the practical diagnosis of M. pneumoniae in clinical specimens. Through comparing 224 sputum specimens, excellent performance of SEA assay with 90.48% sensitivity and 100% specificity relative to real-time PCR was observed. Compared with LAMP, a comparable sensitivity and low false positive rate was observed for SEA method. Therefore, SEA is a promising method for detecting M. pneumoniae directly from clinical specimens, which is especially suitable for point-of-care testing in primary care facilities and resource-limited settings with minimal equipment and technological expertises.

Rapid Detection of the Bursaphelenchus Xylophilus by Denaturation Bubble-mediated Strand Exchange Amplification.

Bursaphelenchus xylophilus (B. xylophilus) is one of the most important causal agents of infectious diseases in forest pathology. Obviously, the rapid detection of B. xylophilus is an urgent need for its prevention and cure. We have developed a detection method of B. xylophilus by strand exchange amplification (SEA). This method could detect 105 copies of genomic DNA of B. xylophilus, and it was sufficiently sensitive to detect a single nematode as short as 40 min. Moreover, because the amplification result could be visualized by the naked eyes, the only equipment required throughout the process was a simple isothermal block. Therefore, our method would be a potential for developing on-site detection of B. xylophilus to prevent and control its spread.

A simple isothermal nucleic acid amplification method for the effective on-site identification for adulteration of pork source in mutton

As meat adulteration issue is becoming increasingly prominent worldwide, it is crucial to realize on-site detection with simple equipment. Conventional nucleic acid amplification methods are reliable but the requirement of complex equipment, skilled technicians and long operation time limit their on-site use. Here, a simple denaturation bubble-mediated strand exchange amplification method (SEA) requiring only a pair of primers and one polymerase was first reported for identifying adulteration of pork source by targeting the specie-specific mitochondrial DNA sequence. The SEA method displayed good specificity for pork and could detect as low as 30 pg/μL pork DNA. In binary mixtures, the SEA method could detect 1% pork meat total DNA by both colorimetric and fluorescence determination, fulfilling the requirement of artificial meat adulteration. Excitedly, the whole detection process could be finished within 1 h by coupling with fast tissue DNA extraction method, only requiring a simple heating block. Therefore, with simplicity and rapidity, SEA method will be suitable for on-site meat species identification.

On-site Method for Beef Detection Based on Strand Exchange Amplification.

Meat screening plays a significant role in human health and religion. But the identification methods for beef were little reported. In this work, a simple colorimetric method based on denaturation bubble-mediated strand exchange amplification (SEA) was developed for the rapid and sensitive identification of beef. The whole strategy was performed on a portable metal bath and the distinguishable color between positive and negative controls was observed directly by the naked eyes. The feasibility using crude extraction samples by a heating treatment in PBS for 2 min was evaluated in duck spiked by beef. The result demonstrated that the developed method could identify as low as 1% (w/w) beef/duck within 50 min. Meanwhile, the results showed the method had a good repeatability and specificity. Therefore, this assay allows for the rapid, sensitive, specific detection of beef, and can be recommended as an effective, promising strategy for on-site meat identification.

Rapid detection of foodborne pathogen Listeria monocytogenes by strand exchange amplification

A strand exchange amplification (SEA) method to detect foodborne pathogen Listeria monocytogenes was developed. SEA is a novel nucleic acid amplification method that only requires one pair of primers. The specie-specific primers were designed by targeting the 16S rRNA gene and the amplification reaction was performed as short as 60 min at 61 °C. Notably, SEA method could not only detect genomic DNA but also detect RNA by one step without requiring extra reverse transcription. The result could be visualized by naked eyes so that water bath pot would be the only equipment needed. Moreover, culture fluids and bacteria colony could be successfully detected without any pretreatment and the method displayed good specificity and strong anti-jamming capacity. These features greatly simplified the operating procedure and made SEA method be potential for developing point-of-care testing (POCT) devices to detect viable L. monocytogenes.

Triggered isothermal PCR by denaturation bubble-mediated strand exchange amplification.

Here, we introduced the concept of strand exchange amplification (SEA) mediated by denaturation bubbles. Similar to traditional PCR, it only employed a DNA polymerase and a pair of common primers to realize a three-step cycle process, but the entire SEA reaction was performed at a single temperature.