Recombinase-aided Amplification Assay Research Articles

A novel photoreactive DNA-binding dye for detecting viable Klebsiella pneumoniae in powdered infant formula

In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA-RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA-RAA assay can detect as low as 2.6 × 103 cfu/mL of K. pneumoniae in pure culture and 2.3 × 104 cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 100 cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA-RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF.

Rapid Detection of Multi-Resistance Strains Carrying mcr-1 Gene Using Recombinase-Aided Amplification Directly on Clinical Samples.

With the increasingly severe problem of bacterial resistance, colistin, as the last line of defense, has attracted attention again. Mobile colistin resistance (mcr-1) gene is involved in the horizontal transmission of colistin resistance in Gram-negative bacteria (GNB), which is a serious threat to human health. Therefore, rapid detection of mcr-1 gene presence in clinical samples is crucial. In this study, a Recombinase-aided amplification(RAA) method for mcr-1 was successfully constructed, with sensitivity of 20 copies/reaction. In addition, amplification signal could only be detected in the strain containing mcr-1 gene among 14 different bacterial species. The method was then used to test a total of 672 clinical samples from a pediatric hospital in Beijing. Five strains harbored mcr-1 genes were isolated from mcr-1-positive clinical samples and identified as Escherichia coli. Multi-locus sequence typing (MLST) analysis showed that the five E. coli belonged to different ST types. Notably, the mcr-1 gene from the isolates could be transferred conjugately to the recipient strain E. coli J53, with highest transfer efficiency up to 57–58%, suggesting that the mcr-1 gene was located on the plasmid. These findings showed that the RAA assay has potential to be a rapid and sensitive mcr-1 gene screening test for clinical samples, and mcr-1 could be transmitted vertically and horizontally between and within bacterial species in a plasmid-mediated manner.

Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral flow dipstick

Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida (P. multocida), Escherichia coli (E. coli), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/μL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.

Development and Application of a Reverse-Transcription Recombinase-Aided Amplification Assay for Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus currently widespread worldwide in the swine industry. Since PEDV was discovered in China in 1984, it has caused huge economic losses in the swine industry. PEDV can infect pigs of all ages, but piglets have the highest infection with a death rate as high as 100%, and the clinical symptoms are watery diarrhea, vomiting, and dehydration. At present, there is not any report on PEDV detection by RT-RAA. In this study, we developed an isothermal amplification technology by using reverse-transcription recombinase-aided amplification assay (RT-RAA) combined with portable instruments to achieve a molecular diagnosis of PEDV in clinical samples from China. By designing a pair of RT-RAA primers and probes based on the PEDV N gene, this method breaks the limitations of existing detection methods. The assay time was within 30 min at 41 °C and can detect as few as 10 copies of PEDV DNA molecules per reaction. Sixty-two clinical tissue samples were detected by RT-qPCR and RT-RAA. The positive and negative rates for the two methods were 24.19% and 75.81%, respectively. Specificity assay showed that the RT-RAA had specifically detected PEDV and was not reactive for porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), swine flu virus (SIV), or porcine Japanese encephalitis virus (JEV). The results suggested that RT-RAA had a strong specificity and high detection sensitivity when combined with a portable instrument to complete the detection under a constant temperature of 30 min, which are more suitable for preventing and controlling PEDV onsite in China.

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus

ObjectiveTo establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene. MethodsA recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay. ResultsThe RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses. ConclusionA highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Development and application of a reverse-transcription recombinase-aided amplification assay for subgroup J Avian leukosis virus

Subgroup J Avian leukosis virus (ALV-J) is an important pathogen of poultry tumor diseases. Since its discovery, it has caused significant economic losses to the poultry industry. Thus, the rapid detection of molecular level with strong specificity is particularly important whether poultry are infected with ALV-J. In this study, we designed primers and probe for real-time fluorescent reverse-transcription recombinase-aided amplification assay (RT-RAA) based on the ALV-J gp85 sequence. We had established a real-time fluorescent RT-RAA method and confirmed this system by verifying the specificity and sensitivity of the primers and probe. In addition, repeatability tests and clinical sample regression tests were used for preliminary evaluation of this detection method. The sensitivity of established method was about 101 copies/μL, and the repeatability of the CV of the CT value is 4%, indicating repeatability is good. Moreover, there was no cross-reactivity with NDV, IBV, IBDV, H9N2, MDV, and REV, and other avian leukosis virus subgroups, such as subgroups A, B, C, D, K and E. Importantly, the real-time fluorescent RT-RAA completed the test within 30 min at a constant temperature of 41°C. Forty-two clinical samples with known background were tested, and the test results were coincided with 100%. Overall, these results suggested that the real-time fluorescent RT-RAA developed in this study had strong specificity, high sensitivity, and good feasibility. The method is simple, easy, and portable, that is suitable for clinical and laboratory diagnosis, and provides technical support for the prevention and control of ALV-J.

Development and evaluation of a sensitive recombinase aided amplification assay for rapid detection of Vibrio parahaemolyticus

Vibrio parahaemolyticus (V. parahaemolyticus) is a widely distributed pathogen in the coastal areas, which causes food poisoning and leads to gastroenteritis and sepsis. Therefore, developing a simple, sensitive, and rapid detection method for V. parahaemolyticus is a major concern globally. This study established a sensitive and rapid technique based on recombinase aided amplification (RAA) to detect V. parahaemolyticus. The RAA reaction was carried out successfully at 39 °C within 30 min. The sensitivity of the RAA assay was 101 copies/μL using the recombinant plasmid and 10−3 ng/μL using the V. parahaemolyticus strain. In addition, RAA directly detected 7 × 103 CFU/mL of simulated fecal samples and 0.1 CFU/mL after enrichment for 4 h. The sensitivity and specificity of the RAA assay using fecal and fish samples were 100% similar to that of the real-time PCR. We conclude that the RAA assay is an ideal screening method for detecting V. parahaemolyticus due to its rapidity, high accuracy, and simplicity in operation.

The fluorescent probe-based recombinase-aided amplification for rapid detection of Escherichia coli O157:H7

Escherichia coli O157:H7 (E. coli O157:H7) is a common foodborne morbigenous microorganism, which can spread through fecal-oral transmission. Humans can be infected by ingesting foods and water contaminated with E. coli O157:H7, which can cause various symptoms. In present study, we have successfully developed a quick and hypersensitive fluorescent probe-based Recombinase-aided amplification (RAA) method and applied in E. coli O157:H7 detection at 39 °C in 20 min. The sensitivity of the assay in pure E. coli O157:H7 suspension was 5.6 × 100 CFU/mL. The fluorescent probe-based RAA assay was further applied in three samples, and the limit of detection (LOD) in skimmed milk, lettuces and lake water was 5.4 × 101 CFU/mL, 7.9 × 101 CFU/mL and 5.2 × 101 CFU/mL, separately. This method showed a high sensitivity and short detection time, which has the feasible application in on-site test in real samples.

Development of a reverse transcription recombinase-aided amplification assay for detection of Getah virus

GETV, an arbo-borne zoonotic virus of the genus Alphavirus, which causes diarrhea and reproduction disorders in swine, lead to serious economic losses to the swine industry in China. At present, the existing methods for GETV detection are time-consuming and low sensitivity, so, a rapid, accurate and sensitive GETV detection method is urgently needed. In this study, a fluorescent reverse transcription recombinase-assisted amplification method (RT-RAA) was successfully established for the rapid detection of GETV. The sensitivity of this method to GETV was 8 copies/reaction and 20 TCID50/reaction. No cross-reaction with other viruses. A total of 118 samples were prepared for GETV detection using fluorescent RT-RAA and SYBR Green I RT-qPCR, the coincidence rate of the two methods was 100%. The results suggest that the RT-RAA method is rapid, sensitive and specific for GETV detection and can be applied in the clinical.

Reverse transcription recombinase-aided amplification assay for rapid detection of the influenza A(H1N1)pdm09 H275Y mutation that confers oseltamivir resistance

The emergence of the influenza A(H1N1)pdm09 virus with the NA-H275Y mutation, which confers oseltamivir resistance, must be monitored, especially in patients undergoing neuraminidase inhibitor treatment. In this study, we developed a reverse transcription recombinase-aided amplification assay that has high sensitivity (detection limit: 1.0 × 101 copies/μL) and specificity for detecting the oseltamivir-resistant H275Y mutation; the assay is performed within 30 min at a constant temperature of 39° Celsius using an isothermal device. This method is suitable for the clinical application of targeted testing, thereby providing technical support for precision medicine in individual drug applications for patients with severe infection or immunosuppression.

A Recombinase Aided Amplification Assay for Rapid Detection of the Klebsiella pneumoniae Carbapenemase Gene and Its Characteristics in Klebsiella pneumoniae

Klebsiella pneumoniae carbapenemase genes (blaKPC) play an important role in carbapenem-resistant Enterobacteriaceae in China. A rapid detection method for blaKPC genes and investigations into the molecular characteristics of blaKPC positive Klebsiella pneumoniae were necessary. In this study, an easy and rapid recombinase aided amplification assay (RAA) for blaKPC was established. This protocol could be completed at 39°C in 15–20 min. The sensitivity of this assay was determined as 48 copies per reaction, and the specificity was 100%. The blaKPC RAA method could be used for clinical diagnosis and epidemiological investigation. Among 801 fecal samples from inpatients, 34 blaKPC positive isolates were identified from each sample, of which 23 isolates were K. pneumoniae. ST11 with blaKPC-2 was the most prevalent type. All these strains were multidrug resistant and carried various virulence genes. Fecal carriage of blaKPC positive carbapenem-resistant K.pneumoniae poses significant challenges for public health control.

Rapid Detection of New Delhi Metallo-β-Lactamase Gene Using Recombinase-Aided Amplification Directly on Clinical Samples From Children.

New Delhi metallo-β-lactamase, a metallo-β-lactamase carbapenemase type, mediates resistance to most β-lactam antibiotics including penicillins, cephalosporins, and carbapenems. Therefore, it is important to detect blaNDM genes in children’s clinical samples as quickly as possible and analyze their characteristics. Here, a recombinase-aided amplification (RAA) assay, which operates in a single one-step reaction tube at 39°C in 5−15 min, was established to target blaNDM genes in children’s clinical samples. The analytical sensitivity of the RAA assay was 20 copies, and the various bacterial types without blaNDM genes did not amplify. This method was used to detect blaNDM genes in 112 children’s stool samples, 10 of which were tested positive by both RAA and standard PCR. To further investigate the characteristics of carbapenem-resistant bacteria carrying blaNDM in children, 15 carbapenem-resistant bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Citrobacter freundii, Klebsiella oxytoca, Acinetobacter junii, and Proteus mirabilis) were isolated from the 10 samples. Notably, more than one bacterial type was isolated from three samples. Most of these isolates were resistant to cephalosporins, cefoperazone-sulbactam, piperacillin-tazobactam, ticarcillin-clavulanic acid, aztreonam, co-trimoxazole, and carbapenems. blaNDM–1 and blaNDM–5 were the two main types in these samples. These data show that the RAA assay has potential to be a sensitive and rapid blaNDM gene screening test for clinical samples. The common existence of blaNDM and multi-drug resistance genes presents major challenges for pediatric treatment.

Rapid Detection of DNA Methylation with a Novel Real-Time Fluorescence Recombinase-Aided Amplification Assay.

Researchers have conducted in-depth research on DNA methylation mechanism, which is related to various diseases such as deficiency of imprinted gene and occurrence of tumors. This study provides a novel rapid quantitative detection assay and real-time fluorescence recombinase-aided amplification assay (RAA) for DNA methylation. Firstly, specific sequence of methylation genes was chosen and primers and fluorogenic probe for RAA experiment were designed and synthesized. Lastly, these amplification products were proven by sequencing and analysis. Results showed that the amplification efficiency and template concentration of RAA had linear dependent (R² > 95%) when the concentration range was 4.64×108 copies/μL˜4.64×10⁴ copies/μL. The test assay can also detect positive samples when the template concentration is below 4.64×10⁴ copies/μL. Remarkably, the entire experiment process only takes 15-20 minutes, so it is beneficial for rapid bedside simple screening of some special DNA methylation sites, such as detection of resistance genes. In a word, this method has very great potential for diseases with DNA methylation in clinical settings, especially if methylation analysis needs to be done quickly and easily.

Performance of a recombinase - aided amplification assay for detection of Schistosoma japonicum infections in Oncomelania hupensis

To evaluate the efficiency of a recombinase-aided amplification (RAA) assay for the detection of Schistosoma japonicum infections in Oncomelania hupensis snails. A group test was employed. Fifty Oncomelania snails were collected as a detection sample. The detection samples without infected snails were designated as negative specimens, while the detection samples that contained different numbers of infected snails were designated as positive specimens. A total of 10 negative specimens, 10 positive specimens containing 1 infected snail, 20 positive specimens containing 2 infected snails and 10 positive specimens containing 3 infected snails were assigned. Following random grouping, 40 specimens were subject to the florescent RAA assay using a blind method. The miradium shedding method served as a gold standard, and the sensitivity, specificity, Youden's index and coincidence rate of the florescent RAA assay were estimated. In addition, 20 samples consisted of 5 negative specimens and 15 positive specimens with 1, 2 and 3 infected snails respectively were grouped randomly. The same specimens were detected using the crushing method and fluorescent RAA assay with the blind method in a paired-design manner. Then, the test results were compared and analyzed. Florescent RAA assay detected 29 positives in the 30 specimens containing different numbers of infected snails, with a sensitivity of 96.67%, and 8 negatives in the 10 detection specimens without infected snails, with a specificity of 80.00%, showing a Youden's index of 0.77. The coincidence rate was 100% among 10 repeated assays for a detection specimen. In addition, there was no significant difference in the detection of infected snails between the florescent RAA assay and the crushing method (χ2 = 0, P > 0.05), and the actual coincidence rates of the florescent RAA assay and crushing method were 95.00% (19/20) and 90.00% (18/20) with the real results, respectively. Fluorescent RAA assay has a favorable efficiency for the detection of S. japonicum infections in Oncomelania snails, which shows a potential in screening of S. japonicum-infected Oncomelania snails.

Development of a recombinase-aided amplification assay for rapid detection of human norovirus GII.4

BackgroundHuman noroviruses are one of the main causes of foodborne illnesses and represent a serious public health concern. Rapid and sensitive assays for human norovirus detection are undoubtedly necessary for clinical diagnosis, especially in regions without more sophisticated equipment.MethodThe rapid reverse transcription recombinase-aided amplification (RT-RAA) is a fast, robust and isothermal nucleic acid detection method based on enzyme reaction. This method can complete the sample detection at 39 °C in 30 min. In this study, we successfully established a rapid reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of human norovirus GII.4 and applied this assay to clinical samples, as well as comparison with commercial reverse transcription real-time fluorescence quantitative PCR (RT-qPCR).ResultsAt 95% probability, the detection sensitivity of RT-RAA was 3.425 log10 genomic copies (LGC)/reaction. Moreover, no cross-reaction was observed with other norovirus genogroups and other common foodborne viruses. Stool samples were examined by RT-RAA and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Compared of RT-qPCR, kappa values for human norovirus detection with RT-RAA were 0.894 (p < 0.001), indicating that both assays were in agreement.ConclusionThis RT-RAA assay provides a rapid, specific, and sensitive assay for human norovirus detection and is suitable for clinical testing.

Field Validation of a Rapid Recombinase Aided Amplification Assay for SARS-CoV-2 RNA at Customs — Zhejiang Province, China, January 2021

IntroductionThe best approach to preventing the importation of coronavirus disease 2019 (COVID-19) is enhancing the detection capacity at customs. The rapid detection is of utmost importance and therefore highly demanded.MethodsWe conducted a field validation study of a duplex real-time reverse transcription recombinase-aided amplification (RT-RAA) assay in Zhoushan and Hangzhou customs, in Zhejiang Province, China. The reverse transcriptase polymerase chain reaction (RT-PCR) assay kit routinely used at customs was used in parallel, and the duration the two methods took to complete a specific number of samples was compared.ResultsAmong 506 samples collected, RT-RAA results were consistent with the RT-PCR results. The sensitivity and specificity were 100%, the total coincidence rate was 100%, and the Kappa value was 1 (P<0.05) for both methods. The RT-RAA kit took a significantly shorter time in testing the 20–200 samples than the RT-PCR kit. DiscussionThe RT-RAA detection method is more efficient and suitable for use at customs than RT-PCR assay to realize rapid customs clearance of 200 or fewer samples.

A reverse-transcription recombinase-aided amplification assay for the rapid detection of N gene of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)

The current outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China firstly. A rapid, highly sensitive, specific, and simple operational method was needed for the detection of SARS-CoV-2. Here, we established a real-time reverse-transcription recombinase-aided amplification assay (RT-RAA) to detect SARS-CoV-2 rapidly. The primers and probe were designed based on the nucleocapsid protein gene (N gene) sequence of SARS-CoV-2. The detection limit was 10 copies per reaction in this assay, which could be conducted within 15 min at a constant temperature (39 °C), without any cross-reactions with other respiratory tract pathogens, such as other coronaviruses. Furthermore, compared with commercial real-time RT-PCR assay, it showed a kappa value of 0.959 (p < 0.001) from 150 clinical specimens. These results indicated that this real-time RT-RAA assay may be a valuable tool for detecting SARS-CoV-2.

Development of a recombinase-aided amplification assay for rapid and sensitive detection of porcine circovirus 3

Porcine circovirus type 3 (PCV3) is a novel member of the genus Circovirus, first detected in the United States in 2016, with subsequent reports in many countries. PCV3 infections have caused serious economic losses in the pig industry. Alternative rapid and sensitive assays for PCV3 detection are needed for clinical diagnosis, especially in laboratories not equipped with more sophisticated equipment. Here, a real-time recombinase-aided amplification assay (RAA) was developed for PCV3 detection. Specific primers and probes targeting the conserved region of the capsid gene of PCV3 were designed. The assay was performed at 39 °C for 30 min using specialized equipment. Furthermore, 36 clinical samples were used to evaluate the RAA. The analytical sensitivity of the RAA for PCV3 was 38 copies per reaction at 95% probability level, using a probit regression model. There was no cross-reactivity with other DNA viruses belonging to the Circoviridae and Parvoviridae families. The detection rate agreed with that obtained by an established real-time PCR assay with a kappa value of 1.0. Our results demonstrated that this new RAA could be used for the rapid, accurate, and sensitive detection of PCV3.

Development of a recombinase-aided amplification assay for detection of orf virus

Orf, caused by orf virus (ORFV), is an important zoonotic disease that infects goat and sheep, leading to huge economic losses. ORFV can also cause cutaneous lesions in people who come in close contact with the diseased animals. Although accurate diagnostic methods for ORFV infection exist, there is a need for a rapid, specific, and sensitive method for easy clinical application. Here, we successfully established a recombinase-aided amplification (RAA) assay for rapid detection of ORFV. The analytical sensitivity of the assay for ORFV detection is 1 × 101 copies per reaction. Moreover, no cross-reaction was observed with other common DNA viruses. A total of 45 archived suspected ORFV infected nasal scab skin samples were examined by RAA and SYBR Green-based real-time polymerase chain reaction (PCR). Compared with the real-time PCR assay, the kappa values of the RAA assay for ORFV detection was 0.845 (p <0.001), indicating that both assay results were fully in agreement. In conclusion, this detection assay provides a rapid, sensitive, and specific method for ORFV detection and is suitable for ORFV clinical testing.

Field applicable detection of hepatitis B virus using internal controlled duplex recombinase-aided amplification assay and lateral flow dipstick assay.

Hepatitis B virus (HBV) is a widespread blood-borne pathogen associated with the complication of liver cirrhosis and hepatocellular carcinoma, particularly in south-east Asian and African countries where HBV is highly endemic and the budget and resources are limited. Therefore, simple, rapid, and portable field detection methods are crucial to efficiently control HBV infection. In this study, using heat-treated DNA, we developed two-field applicable detection assays for HBV based on recombinase-aided amplification (RAA). One was an internal controlled duplex RAA assay using a portable real-time fluorescence detection device, another was an instrument-free visual observation assay using lateral flow dipsticks. The entire experimental time was greatly shortened to less than 40 minutes at 39.0°C. The sensitivities, specificities, and clinical performance of both assays were evaluated. Compared with quantitative polymerase chain reaction assay as a reference, our results demonstrated that the two RAA-based assay obtained 97.18% and 95.77% of sensitivity, respectively, and the specificity was 100%, by testing a total of 157 serum samples with HBsAg positive. We conclude that the advantages of rapidity, simplicity, portability, and visualization of proposed two assays make them great potentials in point-of-care testing of HBV infection by untrained people in resource-limited situations.