An overview of mycetoma and its diagnostic dilemma: Time to move on to advanced techniques

The neglected tropical disease mycetoma can become extremely devastating, and can be caused both by fungi and bacteria; these are popularly known as eumycetoma and actinomycetoma respectively. The classical triad of the disease is subcutaneous swelling, multiple discharging sinuses and the presence of macroscopic granules. The present study aims to highlight the existing diagnostic modalities and the need to incorporate newer and more advanced laboratory techniques like pan fungal/pan bacterial 16S rRNA gene polymerase chain reaction (PCR) and sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). It is important for the medical team to be aware of the various diagnostic options (both existing and future), so that diagnosis of such a debilitating disease is never missed, both by clinicians and microbiologists/pathologists. The newer diagnostic methods discussed in this article will help in rapid, accurate diagnosis thus facilitating early treatment initiation, and decreasing the overall morbidity of the disease. In the Indian context, newer technologies need to be made available more widely. Making clinicians aware and promoting research and development in mycetoma diagnostics is the need of the hour.

Advance in the application of recombinase polymerase amplification-lateral flow strips technology in the diagnosis of pathogenic microorganisms / 中华检验医学杂志

Recombinase polymerase amplification (RPA) is a newly developed isothermal amplification technology with high sensitivity and specificity. The combination of RPA and lateral flow strips (LFS) enables rapid identification of target genes. This technique has been widely used in medicine, food, botany, and other fields. This review generalizes the use of RPA-LFS technology for the diagnosing pathogenic microorganisms, providing a reference for point-of-care diagnosis of pathogenic microorganisms.

Research progress of new isothermal amplification technology in rapid diagnosis of Plasmodium and other parasites / 中国热带医学

@#Abstract: At present, nucleic acid detection technology based on the PCR principle is commonly used to detect malaria parasites, the existing Plasmodium detection methods mainly include microscopy, antigen immunoassay, and nucleic acid detection,but due to the long detection time, high personnel and equipment requirements, and other shortcomings, its popularization, and application at the grassroots level are limited. What challenges previous Plasmodium detection methods are the lack of experienced professionals and advanced equipment at the grassroots as well as the requirement of rapid detection of large samples under extreme conditions. The isothermal amplification technology developed in recent years has potential application prospects due to its simplicity, rapidity, high sensitivity, and high specificity. This article attempts to review the principles, characteristics, and prospects of various isothermal amplification technologies, and on this basis, focuses on the introduction of recombinase polymerase amplification (RPA) and recombinase⁃aided isothermal amplification (RAA) assay technologies and proposes the use of such recombinant enzyme amplification technologies to achieve rapid and accurate diagnosis of common Plasmodium species possibility and imagination.

Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification / 生物医学与环境科学（英文）

Objective@#To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) .@*Methods@#We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD).@*Results@#CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 -18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 2 CFU/g.@*Conclusion@#The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.

Principle of recombinase polymerase amplification and its application progress in medical laboratory science / 中华检验医学杂志

Recombinase polymerase amplification (RPA) is a novel technology for nucleic acid isothermal amplification. It can achieve the rapid amplification and detection of a target gene under 37-42 ℃. This amplification method is highly sensitive, more specific and less instrument-dependent than other existing methods, and it can also integrate multiple detection modes. Therefore, it is especially suitable for applying in low-resource settings and conducting point-of-care tests. Starting from the reaction principles and the experimental design of RPA, this article pointed out some key points when using RPA in a clinical setting. The current development and related problems of RPA were concluded and the various future uses of this method were also prospected.

Detection methods and devices of visual detection in closed tube recombinase polymerase amplification of hepatitis B virus / 医学研究生学报

ObjectiveThe traditional detection method of recombinase polymerase amplificaton (RPA) is not suitable for rapid field detection due to the complicated operation and other factors. Taking the detection of hepatitis b virus (HBV) nucleic acid as an example, it established a detection method of HBV nucleic acid isothermal amplification based on recombinase polymerase amplificaton (RPA) and designed a matching visual detection device of RPA product.MethodsFirstly, a RPA product detection device was designed, which can be used to collect images by taking photos of mobile phones and visually interpret the detection results. Secondly, RPA primers and probes were designed according to the design of HBV gene conserved sequence. Amplification efficiency of each primer pairs were compared though monitoring the RPA reaction of real-time fluorescence curve to screen the best primers and optimize the optimal reaction conditions. Visual detection sensitivity was investigated by using artificial synthesis of HBV target plasmid, and was investigated the specificity of the method by the detection of synthetic plasmid containing hepatitis c virus (HCV), human immunodeficiency virus, treponema pallidum, influenza virus, human papilloma virus DNA fragment. Thirdly, the feasibility of RPA product visualization detection device was verified by comparing with the real-time fluorescence amplification curve. Finally, RPA visual detection was performed on 20 serum DNA samples detected by real-time fluorescence PCR to evaluate the applicability of this method to the detection of actual clinical samples.ResultsThe visual detection device of RPA product was used to realize the negative and positive signals that could be detected by mobile phone photography and visual observation. The visual detection method of HBV nucleic acid RPA amplification could realize the visual detection of DNA targets as low as 1-10 copies of HBV within 30 min at 39 ℃ and had good specificity. The test results of 20 serum DNA samples were completely consistent with those of the commercially available qPCR kit, which preliminarily verified the practicability of the method and the device.ConclusionCombined the established HBV-RPA amplification system with the RPA product visualized detection device, it would be expected to develop a low-cost rapid visualization screening technology platform for HBV nucleic acid in blood.

Preliminary application of real-time fluorescence recombinase polymerase amplification in Candida albicans / 中华烧伤杂志

Objective@#To explore the preliminary application effect of real-time fluorescence recombinase polymerase amplification (RPA) in the detection of Candida albicans.@*Methods@#(1) Candida albicans standard strain and negative control bacteria of Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Candida glabrata standard strains of respectively 1 mL were collected and their DNA were extracted by yeast/bacterial genomic kit. The specificity of polymerase chain reaction (PCR), real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting Candida albicans were analyzed. (2) One Candida albicans standard strain and one negative control bacteria of Candida glabrata standard strain were collected, resuscitated, and counted. Candida albicans was diluted 10 times to 1×107 to 1×101 colony-forming unit (CFU)/mL. The DNA of the two bacteria were extracted as experiment (1). The sensitivity of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting Candida albicans were analyzed. The number of cycles for amplification curve to reach the threshold in real-time fluorescent quantitative PCR, and time of appearance of specific amplification curve in real-time fluorescence RPA were recorded and compared with the results in PCR. The detection limit and rate of the above-mentioned 3 methods in detecting Candida albicans were analyzed, and the correlation between concentration of Candida albicans in real-time fluorescence RPA and detection time was analyzed. (3) One standard strain of Candida albicans was collected, and the DNA was extracted as experiment (1) and detected by PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA. The total detection time of the above-mentioned 3 methods was recorded, respectively. (4) The DNA of 31 clinical samples of suspected Candida albicans infection and 1 clinical sample of Candida albicans collected from cotton swab were extracted, PCR and real-time fluorescence RPA were carried out, and the positive detection rates of the above-mentioned methods were calculated. The DNA of the clinical samples with positive results in both PCR and real-time fluorescence RPA were extracted by yeast/bacterial genomic kit, chelex-100 boiling method, and repeatedly freeze-thawing with liquid nitrogen method, and real-time fluorescence RPA and PCR were carried out. The negative control bacteria was Candida glabrata in real-time fluorescence RPA, while negative control bacteria in PCR were the same as experiment (1). The positive results in PCR and real-time fluorescence RPA were observed and time for amplification curve to reach the fluorescence threshold in real-time fluorescence RPA was recorded, respectively. Data were processed with linear correlation analysis and t test.@*Results@#(1) Three methods showed positive results in detecting standard strain of Candida albicans, and none of the 5 negative control bacteria showed positive results. (2) As the concentration of bacterial solution of Candida albicans decreased, the number of cycles for the amplification curve to reach the threshold increased in real-time fluorescent quantitative PCR, the time for appearance of specific amplification curve prolonged in real-time fluorescence RPA, and brightness of the gel strip weakened in PCR. None of the negative control bacteria in the above-mentioned 3 detection methods showed corresponding positive results. The detection limit of Candida albicans in real-time fluorescence RPA, PCR, and real-time fluorescent quantitative PCR was 1×101 CFU/mL. There was a significant negative correlation between the concentration of Candida albicans and the detection time in real-time fluorescence RPA (r=-0.95, P<0.01). The positive detection rates of PCR and real-time fluorescent quantitative PCR for Candida albicans of 1×101 to 1×107 CFU/mL were 100%. The positive detection rate of real-time fluorescence RPA for Candida albicans of 1×101 CFU/mL was 78%, and the positive detection rate of real-time fluorescence RPA for Candida albicans of 1×102 to 1×107 CFU/mL was 100%. (3) The total time of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA detection for Candida albicans was 133, 93, and 35 min, respectively. (4) The positive detection rate of real-time fluorescence RPA for 31 clinical samples of suspected Candida albicans infection was 32.26% (10/31), which was slightly lower than 35.48% (11/31) of PCR. Eleven clinical samples showed positive results both in real-time fluorescence RPA and PCR detection. No positive result was observed in the negative control bacteria detected both by real-time fluorescence RPA and PCR. The DNA was extracted by yeast/bacterial genomic extraction kit and chelex-100 boiling method for real-time fluorescence RPA detection. The time for the amplification curve to reach the threshold was (438±13) and (462±12) s, respectively, which was close (t=1.32, P>0.05). The DNA was extracted by repeatedly freeze-thawing with liquid nitrogen method for real-time fluorescence RPA, and the time for the amplification curve to reach the threshold in real-time fluorescence RPA was (584±15) s, which was significantly longer than that in the other 2 methods (t=7.55, 6.39, P<0.01).@*Conclusions@#Real-time fluorescence RPA has advantages of rapid detection, simple operation, high sensitivity, and good specificity in detecting Candida albicans, which is worthy of clinical application.

Application of NASBA and RPA in detection of pathogenic bacteria / 中华流行病学杂志

Nucleic acid sequence-based amplification and recombinase polymerase amplification are the recently developed thermostatic amplification techniques based on PCR. This paper briefly summarizes the principle of reaction, design principle of primer and probe, advantage of these two techniques (simple, accurate, highly sensitive and rapid) and introduces the application of the techniques in the detection of pathogenic bacteria.

Exploratory research on detection of influenza viruses by reverse transcriptase-recombinase polymerase amplification / 中华实验和临床病毒学杂志

Objective@#To establish and hevaluate a detection method for influenza virus using reverse transcriptase-recombinase polymerase amplification (RT-RPA).@*Methods@#RT-RPA was developed for detection of influenza viruses (type A and B) and subtyping of H1 and H3 using the primers targeted matrix and hemagglutinin (HA) genes of influenza A virus, and non-structural (NS) protein gene of influenza B virus. The specificity and sensitivity of RT-RPA were determined.@*Results@#The RT-RPA for the detection of influenza viruses showed specific amplification products of corresponding target gene, but no amplification products for other respiratory viruses, indicating that the method had good specificity. The detection limits of RT-RPA were 100 copies/μl. RT-RPA combined with SYBR Green I was used for the detection of influenza B virus with the detection limit of 100 copies/μl.@*Conclusions@#The feasibility of detecting influenza virus by RT-RPA was preliminarily confirmed.

Real-time recombinase polymerase amplification for detection of Vibrio parahaemolyticus / 预防医学

Objective@# To establish real-time recombinase polymerase amplification（RPA）for the rapid detection of Vibrio parahaemolyticus（VP）.@*Methods@#An exo probe and primers were designed according to the conserved sequence of thermolabile hemolysin（tlh）gene of VP and then RPA for detection of VP was established. The sensitivity of the assay was evaluated by detecting different concentration of VP;the specificity was evaluated by detecting different bacteria;the stability was evaluated by repeat trials;the application effect was evaluated by detecting food samples which were simultaneously tested with traditional culture method according to GB 4798.7-2013 Detection of VP.@*Results@#A real-time RPA was established to complete VP amplification within 20 min at a constant temperature of 39 ℃. The analytical sensitivity of the assay was five pg per reaction and no cross-reactivity with other pathogenic bacteria observed. The RPA detection results with different concentration of VP and E. coli DNA templates at three time points were consistent. The detection results of 51 food samples by RPA were the same as those by traditional culture method.@*Conclusion@#The established real-time RPA can qualitatively detect VP,with simple operation and interpretation of results,which is suitable for rapid detection of VP in public health emergencies and food safety supervision.

Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick

Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39℃ with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.

Application of recombinase polymerase amplification in the detection of Pseudomonas aeruginosa / 中华烧伤杂志

Objective@#To establish an optimized method of recombinase polymerase amplification (RPA) to rapidly detect Pseudomonas aeruginosa in clinic.@*Methods@#(1) The DNA templates of one standard Pseudomonas aeruginosa strain was extracted and detected by polymerase chain reaction (PCR), real-time fluorescence quantitative PCR and RPA. Time of sample loading, time of amplification, and time of detection of the three methods were recorded. (2) One standard Pseudomonas aeruginosa strain was diluted in 7 concentrations of 1×107, 1×106, 1×105, 1×104, 1×103, 1×102, and 1×101 colony forming unit (CFU)/mL after recovery and cultivation. The DNA templates of Pseudomonas aeruginosa and negative control strain Pseudomonas putida were extracted and detected by PCR, real-time fluorescence quantitative PCR, and RPA separately. The sensitivity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (3) The DNA templates of one standard Pseudomonas aeruginosa strain and four negative control strains (Staphylococcus aureus, Acinetobacter baumanii, Candida albicans, and Pseudomonas putida) were extracted separately, and then they were detected by PCR, real-time fluorescence quantitative PCR, and RPA. The specificity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (4) The DNA templates of 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin, 1 clinical strain of which was taken by cotton swab, and negative control strain Pseudomonas putida were extracted separately, and then they were detected by RPA. Positive amplification signals of the clinical strains were observed, and the detection rate was calculated. All experiments were repeated for 3 times. Sensitivity results were analyzed by GraphPad Prism 5.01 statistical software.@*Results@#(1) The loading time of RPA, PCR, and real-time fluorescence quantitative PCR for detecting Pseudomonas aeruginosa were all 20 minutes. In PCR, time of amplification was 98 minutes, time of gel detection was 20 minutes, and the total time was 138 minutes. In real-time fluorescence quantitative PCR, amplification and detection could be completed simultaneously, which took 90 minutes, and the total time was 110 minutes. In RPA, amplification and detection could also be completed simultaneously, which took 15 minutes, and the total time was 35 minutes. (2) Pseudomonas putida did not show positive amplification signals or gel positive results in any of the three detection methods. The detection limit of Pseudomonas aeruginosa in real-time fluorescence quantitative PCR and PCR was 1×101 CFU/mL, and that of Pseudomonas aeruginosa in RPA was 1×102 CFU/mL. In RPA and real-time fluorescence quantitative PCR, the higher the concentration of Pseudomonas aeruginosa, the shorter threshold time and smaller the number of cycles, namely shorter time for detecting the positive amplified signal. In real-time fluorescence quantitative PCR, all positive amplification signal could be detected when the concentration of Pseudomonas aeruginosa was 1×101-1×107 CFU/mL. In RPA, the detection rate of positive amplification signal was 0 when the concentration of Pseudomonas aeruginosa was 1×101 CFU/mL, while the detection rate of positive amplification signal was 67% when the concentration of Pseudomonas aeruginosa was 1×102 CFU/mL, and the detection rate of positive amplification signal was 100% when the concentration of Pseudomonas aeruginosa was 1×103-1×107 CFU/mL. (3) In RPA, PCR, and real-time fluorescence quantitative PCR, Pseudomonas aeruginosa showed positive amplification signals and gel positive results, but there were no positive amplification signals or gel positive results in four negative control strains of Acinetobacter baumannii, Staphylococcus aureus, Candida albicans, and Pseudomonas putida. (4) In RPA, 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin and 1 clinical strain of Pseudomonas aeruginosa taken by cotton swab showed positive amplification signals, while Pseudomonas putida did not show positive amplification signal. The detection rate of positive amplification signal of 29 clinical strains of Pseudomonas aeruginosa in RPA was 100%.@*Conclusions@#The established optimized RPA technology for fast detection of Pseudomonas aeruginosa requires shorter time, with high sensitivity and specificity. It was of great value in fast detection of Pseudomonas aeruginosa infection in clinic.

Rapid detection of human adenovirus by recombinase polymerase amplification assay and lateral flow dipstick / 中华实验和临床病毒学杂志

Objective@#To establish a rapid and sensitive isothermal amplification assay for the detection of human Adenovirus.@*Methods@#Primers and probe used for recombinase polymerase amplification(RPA)were designed based on the conserved region of the adenoviruses hexon gene. After optimizing the reaction temperature and times, the products of RPA were detected by capillary electrophoresis and lateral flow dipstick(LFD). Sensitivity and specicity of the assay were evaluated. The diagnostic value of the RPA-LFD assay was verified using clinical samples which were simultaneously tested by real time PCR assay.@*Results@#The analytical sensitivity of RPA-LFD assay was 2 copies DNA molecules per reaction and no cross reaction with other pathogens was observed. Compared with real-time PCR assay, the sensitivity, and specificity of the present assay were all 100%.@*Conclusions@#The RPA-LFD assay developed in this study has the characteristics of high specificity, sensitivity, rapid and no requirement of expensive equipment which provided a new tool for rapid detection of human adenovirus.

Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of adenovirus / 军事医学

Objective To develop a rapid, accurate, visual, and portable detection method for adenovirus types B (AdvB) and E ( AdvE).Methods Universal primers were targeted on type-specific conserved regions to allow the simultaneous detection of both human Adv (HAdV) species.A detection method based on the combination of recombinase polymerase amplification ( RPA) and lateral flow dipstick ( LFD) was established the sensitivity and specificity evaluated , and throat swab specimens of 19 patients infected with AdvB and AdvE as well as 10 healthy volunteers were detected with this method.Results The detection limit of the method was 10 copies/μl Adv DNA, which was close to that of qPCR , and there were no cross-reactions with other species of Adv and unrelated virus .The detection could be finished within 15 to 20 min within the temperature range of 25 to 45℃.When applied to clinical samples , this method showed 100% sensitivity and specificity.Conclusion This detection assay is a sensitive , specific, rapid and simple method that eliminates the need for expensive equipment , trained personnel or laboratories .The characteristics of this system render it suitable for use in grass-roots healthcare departments , and the system is especially effective for field testing and on-site testing.

Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of adenovirus / 军事医学

Objective To develop a rapid, accurate, visual, and portable detection method for adenovirus types B (AdvB) and E ( AdvE).Methods Universal primers were targeted on type-specific conserved regions to allow the simultaneous detection of both human Adv (HAdV) species.A detection method based on the combination of recombinase polymerase amplification ( RPA) and lateral flow dipstick ( LFD) was established the sensitivity and specificity evaluated , and throat swab specimens of 19 patients infected with AdvB and AdvE as well as 10 healthy volunteers were detected with this method.Results The detection limit of the method was 10 copies/μl Adv DNA, which was close to that of qPCR , and there were no cross-reactions with other species of Adv and unrelated virus .The detection could be finished within 15 to 20 min within the temperature range of 25 to 45℃.When applied to clinical samples , this method showed 100% sensitivity and specificity.Conclusion This detection assay is a sensitive , specific, rapid and simple method that eliminates the need for expensive equipment , trained personnel or laboratories .The characteristics of this system render it suitable for use in grass-roots healthcare departments , and the system is especially effective for field testing and on-site testing.

Advances in recombinase polymerase amplification / 军事医学

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification technology first re-ported in 2006 by Piepenburg et al.This technology has been shown to typically work at temperatures ranging from 25 to 43℃and can detect products within 5-20 min.RPA technology requires little instrumentation for the nucleic acid amplifi-cation reaction and can be performed not only in PCR tubes , but also in simple devices′such as paper .Combined with probe-based detection methods or lateral flow dipstick assay , it can perform quantitative or visual detection respectively . RPA is a technology that is potentially ideal for point-of-care diagnosis and disease prevention and control ,characterized by high sensitivity, high efficiency, high specificity and user-friendliness.This paper introduces the advantages and develop-ment of RPA technology in reaction conditions and product detection ,summarizes the current applications of this technolo-gy,and predicts the trend of application of RPA technology in point-of-care diagnosis and disease prevention and control .