Amplification Method For Detection Research Articles

Comparison of targeted next-generation sequencing and the Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis in clinical isolates and sputum specimens.

Targeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of many genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and another molecular testing tool, Xpert MTB/rifampicin (RIF), have been empirical. Here, using a dilution series of a RIF-resistant clinical isolate of MTB, we found that tNGS had a slightly lower limit of bacterial detection (102 CFU/mL) compared with Xpert MTB/RIF (103 CFU/mL) in culture medium. However, the minimum detection limit of the rpoB S450L mutation in this isolate was significantly lower with tNGS (102 CFU/mL) than with Xpert MTB/RIF (106 CFU/mL). Sputum samples collected from 129 suspected pulmonary tuberculosis patients were also prospectively studied with the clinical diagnosis as a reference, revealing that the sensitivity of tNGS (48.6%) was higher than those of culture (46.8%), Xpert MTB/RIF (39.4%), and smear microscopy (34.9%) testing. Notably, AMR analysis of 56 MTB-positive samples as determined by tNGS revealed high mutation frequencies of 96.4%, 35.7%, 26.8%, and 19.6% in the following AMR-associated genes: rrs, rpoB, katG, and pncA, respectively. The findings of this study provide theoretical support for the differential clinical application of tNGS and Xpert MTB/RIF and suggest that tNGS has greater application value in tuberculosis drug resistance monitoring and prevention.IMPORTANCETargeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and Xpert MTB/rifampicin (RIF) have been empirical. The Xpert MTB/RIF assay is a commercial system that uses the nucleic acid amplification detection method for rapid (2 hours) diagnosis of tuberculosis (TB). The cost of the tNGS and Xpert MTB/RIF assays in this study was similar, at USD 98 and USD 70-104 per sample, respectively, but the time required for tNGS (3 days) was much longer than that required for the Xpert MTB/RIF assay. However, tNGS yielded more accurate results and a larger number of AMR-associated gene mutations, which compensated for the extra time and highlighted the greater application value of tNGS in TB drug resistance monitoring and prevention.

Rapid Visual Detection of Spiroplasma eriocheiris by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye.

In recent years, a new type of Spiroplasma has been found that can cause "tremor disease" of the Chinese mitten crab Eriocheir sinensis. The outbreak of epidemic tremor disease has caused a serious setback in the Chinese mitten crab farming industry, with an incidence rate of more than 30% and mortality rates of 80-100%. Therefore, finding a sensitive method to detect tremor disease in E. sinensis has become a current research focus. In this research, a loop-mediated isothermal amplification detection method coupled with hydroxynaphthol blue dye (LAMP-HNB) was developed and used to rapidly detect Spiroplasma eriocheiris. First, we designed and synthesized specific outer primers, inner primers and loop primers based on the 16S ribosomal RNA gene of S. eriocheiris. Second, the LAMP-HNB detection method for S. eriocheiris was successfully established by screening the primers, adjusting the temperature and time of the reaction, and optimizing the concentrations of Mg2+ and dNTPs. In the specific tests, only samples infected with S. eriocheiris showed positive results, and other infections caused by bacteria and parasites tested negative, proving that the test has high specificity. Moreover, the detection limit was 2.5 × 10-6ng/µL, indicating high sensitivity. This method for detecting S. eriocheiris provides rapid visual output based on LAMP-HNB detection and is a simple, fast, sensitive, and inexpensive method that can be applied to a wide range of field investigations.

Establishment of a recombinase polymerase amplification detection method for Puccinia striiformis f. sp. tritici

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is an airborne disease that endangers wheat during its entire growth period. In this study, the Pst134EA_003354 uncharacterized protein (GenBank: XM_047941824.1) of Pst was used as the target sequence, and the primers PS-RPA-F and PS-RPA-R, as well as the probe PS-LF-probe, were designed for recombinase polymerase amplification (RPA) technology. Flow chromatography was combined with the process to establish an RPA detection method for Pst. This method successfully established visual detection within 10 min under a constant temperature of 39 °C, and the detection results were consistent with those of ordinary PCR analysis. However, it only had high specificity for Pst, and the detection limit was 10 fg/μL. In addition, this rapid method successfully detected Pst from wheat leaves during the field incubation period, indicating substantial benefits for applied use. In summary, the RPA detection method established in this study has the favourable characteristics of high efficiency, simple functionality, and rapid and universal practicability, providing a theoretical basis for the early detection and prevention of Pst.

RNase H2 triggered visual loop-mediated isothermal amplification combining smartphone assisted all-in-one aptamer magnetic enrichment device for ultrasensitive culture-independent detection of Salmonella Typhimurium in chicken meat

Pre-enrichment cultural continues to remain time-consuming and labor-intensive in the detection of low-concentration bacteria in meat samples. Culture-independent detection method is urgently needed in POCT technology. In order to be able to proceed with culture-independent pre-enrichment for low-concentration pathogen detection, a RNase H2 enzyme triggered all-in-one device-based visual Loop-mediated Isothermal Amplification detection method was developed, combining with Salmonella Typhimurium specific Aptamer Magnetic Enrichment steps, termed as the AME-LAMP method. In order to better enable the AME-LAMP method to perform on-site culture-independent detection, a palm-held all-in-one device (PAD) was designed and manufactured. Further, a self-developed smart phone fluorescence analysis app could complete end-point detection, thus eliminating miscalculation. RNase H2 Enzyme triggered AME-LAMP method could detect Salmonella as low as 5.5 CFU/mL while conventional LAMP only detected 550 CFU/mL. By verifying the detection performance of AME-LAMP methods in chicken meat samples, AME-LAMP shows the potential to achieve ultrasensitive Salmonella detection by comparing with ISO isolation method and normal LAMP. With the assistance of smart phone-based PAD, it can be reliably applied for the on-site culture-independent detection of Salmonella Typhimurium.

Establishment of a reverse transcription recombinase-aided amplification detection method for porcine group a rotavirus.

Porcine rotavirus type A (PoRVA) is the main cause of dehydration and diarrhea in piglets, which has a great impact on the development of the pig industry worldwide. A rapid, accurate and sensitive detection method is conducive to the monitoring, control, and removal of PoRVA. In this study, a PoRVA real-time fluorescent reverse transcription recombinase-aided amplification (RT-RAA) assay was developed. Based on the PoRVA VP6 gene, specific primers and probes were designed and synthesized. The sensitivity of RT-RAA and TaqMan probe-based RT-qPCR was 7 copies per reaction and 5 copies per reaction, respectively. The sensitivity of the RT-RAA method was close to TaqMan probe-based RT-qPCR. The detection results of RT-RAA and TaqMan probe-based quantitative real-time RT-PCR methods were completely consistent in 241 clinical samples. Therefore, we successfully established a rapid and specific RT-RAA diagnostic method for PoRVA.

A direct and multiplex digital PCR chip for EGFR mutation

Digital PCR is a sensitive detection method, which has important applicability in liquid biopsy through the measurement of ctDNA. However, the current sample pre-processing of ctDNA and the multiplex detection capability of digital PCR have limitations. In view of the above two aspects, we developed a digital PCR chip with multiplex capability and established a direct amplification detection method without nucleic acid extraction. Through the design and processing of the chip, we established a self-priming multiplex digital PCR chip, which can detect 4 targets using single fluorescence. This method can be applied to most digital PCR chips. In addition, we used the plasma of lung cancer patients to establish a direct digital PCR detection method based on the chip, thereby avoiding disadvantages caused by the ctDNA extraction process. As a proof of concept, we prepared blood plasma samples with different concentration of ctDNA to prove the chip's multiplex detection capabilities and the results suggested that this multiplex digital PCR is accurate. Overall, our platform provides a novel and promising option for the detection of ctDNA.

Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs.

The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.

Characteristics of Isolates of Pseudomonas aeruginosa and Serratia marcescens Associated With Post-harvest Fuzi (Aconitum carmichaelii) Rot and Their Novel Loop-Mediated Isothermal Amplification Detection Methods.

Fuzi (the lateral root of Aconitum carmichaelii Debx.) is a traditional Chinese medicine that is cultivated in more than eight provinces in China. However, it can be easily devastated by post-harvest rot, causing huge losses. Therefore, it is extremely important that the primary causal pathogens of post-harvest Fuzi rot are identified and appropriate detection methods for them are developed to prevent and control losses. In this study, two bacterial strains (X1 and X2) were isolated from rotten post-harvest Fuzi. Based on their morphological, physiological, and biochemical characteristics, housekeeping gene homologies, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) results, these isolates were identified as Pseudomonas aeruginosa and Serratia marcescens. The pathogenicities of these isolates were confirmed by fulfilling Koch’s postulates demonstrating that they were post-harvest Fuzi rot pathogens. Two loop-mediated isothermal amplification (LAMP) methods targeting the gyrase B subunit (gyrB) gene of P. aeruginosa and the phosphatidylinositol glycan C (pigC) gene of S. marcescens were successfully developed, and it was found that the target genes were highly specific to the two pathogens. These LAMP methods were used to detect P. aeruginosa and S. marcescens in 46 naturally occurring Fuzi and their associated rhizosphere soil samples of unknown etiology. The two bacterial assays were positive in some healthy and rotten samples and could be accomplished within 1 h at 65°C without the need for complicated, expensive instruments. To our knowledge, this is the first report of P. aeruginosa and S. marcescens causing post-harvest Fuzi rot. The newly developed methods are expected to have applications in point-of-care testing for the two pathogens under different Fuzi planting procedures and will significantly contribute to the control and prevention of Fuzi rot.

Comparative Study on DNA Amplification Methods for Detection of Carbapenem-Resistant Enterobacteriaceae (CRE)

Background: Carbapenem-resistant Enterobacteriaceae (CRE) is a resistant group of gram-negative bacteria that produces carbapenemase destroying carbapenem molecules causing drug resistances. Among them, the incidence of New Delhi metallo-beta-lactamases (blaNDM-1), a metallocarbapenemases or class B carbapenemases or metallo-β-lactamases (MBL), are increasing worldwide. Objective: To focused on comparative study on DNA amplification methods for detection of CRE in terms of analytical sensitivity and specificity. Materials and Methods: CRE strains (Escherichia coli, Escherichia cloacae, Citrobacter freudii, and Klebsiella pneumonia) were collected from HRH Maha Chakri Sirindhorn Medical Center, Srinakharinwirot University, Nakhon Nayok, Thailand. All specimens were initially screened for CRE isolates by using carbapenem disks inhibition test. The polymerase chain reaction (PCR), loop mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA) assays were further employed for identification of drug resistance blaNDM-1 gene among CRE isolates. The analytical sensitivity and specificity of the three amplification assays were compared and analyzed. Results: The analytical sensitivity test of PCR, LAMP and RPA assays revealed that the limit of detection were 0.74 ng/μL, 7.4 pg/μL, and 0.74 ng/μL, respectively. Specificity test demonstrated that all three assays showed no cross hybridization to the other 15 related bacterial isolates. Conclusion: The data pointed out that LAMP assay was 100 times more sensitive than PCR and RPA, which could be suitable for application as early detection test for carbapenem resistant bacteria. Hence, the physician can select the proper antibiotics to treat the CRE infectious bacteria, which can reduce morbidity and mortality rates in patients. In the future, LAMP will be combined with lateral flow dipstick (LFD) to improve the efficacy of the assay as not only the early detection test with high sensitivity and specificity for carbapenem resistant bacteria, but also as the convenient and rapid screening test. Keywords: Enterobacteriaceae; Carbapenem; Carbapenem-resistant Enterobacteriaceae (CRE); New Delhi metallo-beta-lactamase (blaNDM-1)

Preliminary screening of Salmonella in oral drugs by visual LAMP technology

Objective According to the Chinese Pharmacopoeia, Salmonella should not be detected in oral drugs. In this experiment, LAMP technology was used to detect Salmonella quickly, and the detection results were visible to the naked eye with calcein-Mn2+ dye, so as to provide pharmaceutical companies with the initial screening of drugs containing Salmonella. Methods Four primers (FIP, BiP, F3, B3) were designed to detect Salmonella by loop mediated isothermal amplification using inv A gene of Salmonella, and then the loop mediated isothermal amplification detection method was established. To verify the specificity of the method, LAMP detection was carried out on 10 kinds of laboratory preserved strains. The sensitivity of the method was obtained by detecting 10 times gradient dilution of Salmonella bacteria solution. Finally, the LAMP system of Salmonella was used to detect the oral drugs sold on the market and not tested by the Drug Administration, and the sensitivity of the system in the drug was determined. Based on these methods, Calcein-Mn2+ dye was added to test the availability of the dye. Results After optimization, the specificity of the system was good. For 10 different strains of LAMP detection, only Salmonella appeared trapezoidal band, and other strains did not detect the band; Through the experimental verification, the sensitivity of the system was 3.2 × 101 CFU/mL; The system was used for the oral drugs sold in the market and not passed the inspection of the drug administration The results of LAMP detection showed that no Salmonella was detected. However, the results of LAMP detection showed that there were trapezoidal bands in the test results. The sensitivity of the system in the detection of Salmonella in oral drugs could reach 3.8 × 101 CFU/mL. Adding calcein-Mn2+ dye into the system, the presence or absence of Salmonella can be distinguished by naked eye observation, and the detection limit is about 102 CFU/mL. Conclusion The visual LAMP detection system for Salmonella can be used for the preliminary screening of oral drugs containing Salmonella.

Preliminary screening method of Escherichia coli in oral drugs by visual LAMP technology

According to Chinese Pharmacopoeia, Escherichia coli should not be detected in oral drugs. In this study, a visual LAMP method for rapid detection of Escherichia coli was developed to replace the traditional microbial limit test method. Methods Four specific primers (FIP, BiP, F3, B3) for loop mediated isothermal amplification were designed by using uidA gene of Escherichia coli, and calcein-Mn2+ dye was added to the system to establish a visual loop mediated isothermal amplification detection method for Escherichia coli Finally, the visual LAMP system was used to detect the presence of E.coli and the sensitivity of the system in drugs. Results The specificity of the optimized system was good, using this system to detect 10 different strains, the results showed that only Escherichia coli genome appeared, the system turned green, the other systems were brown yellow; the detection limit of gradient dilution Escherichia coli was 102 CFU/mL by LAMP. Visual LAMP was used to detect the oral drugs sold in the market and not passed the test of the drug administration, and no E.coli was detected, but the oral drugs that were artificially contaminated with E.coli were visualized by LAMP. The color detection limit of the system in oral drugs was 1.8×102 CFU/mL. Conclusion The visual LAMP detection system can be used for the preliminary detection of oral drugs containing Escherichia coli.

Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue.

Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional spatial dimension to investigate multiplexed gene expression, cell types, disease architecture or even data driven discoveries. In situ sequencing (ISS) method based on padlock probes and rolling circle amplification has been used to spatially resolve gene transcripts in tissue sections of various origins. Here, we describe the next iteration of ISS, HybISS, hybridization-based in situ sequencing. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes for high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. HybISS design allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a targeted amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.

Rapid Detection Methods for Bacterial Pathogens in Ambient Waters at the Point of Sample Collection: A Brief Review.

The world is currently facing a serious health burden of waterborne diseases, including diarrhea, gastrointestinal diseases, and systemic illnesses. The control of these infectious diseases ultimately depends on the access to safe drinking water, properly managed sanitation, and hygiene practices. Therefore, ultrasensitive, rapid, and specific monitoring platforms for bacterial pathogens in ambient waters at the point of sample collection are urgently needed. We conducted a literature review on state-of-the-art research of rapid in-field aquatic bacteria detection methods, including cell-based methods, nucleic acid amplification detection methods, and biosensors. The detection performance, the advantages, and the disadvantages of the technologies are critically discussed. We envision that promising monitoring approaches should be automated, real-time, and target-multiplexed, thus allowing comprehensive evaluation of exposure risks attributable to waterborne pathogens and even emerging microbial contaminants such as antibiotic resistance genes, which leads to better protection of public health.

Development of a Portable SPR Sensor for Nucleic Acid Detection.

Nucleic acid detection is of great significance in clinical diagnosis, environmental monitoring and food safety. Compared with the traditional nucleic acid amplification detection method, surface plasmon resonance (SPR) sensing technology has the advantages of being label-free, having simple operation, and providing real-time detection. However, the angle scanning system in many SPR angle modulation detection applications usually requires a high-resolution stepper motor and complex mechanical structure to adjust the angle. In this paper, a portable multi-angle scanning SPR sensor was designed. The sensor only uses one stepping motor to rotate a belt, and the belt pulls the mechanical linkages of incident light and reflected light to move in opposite directions for achieving the SPR angle scanning mode that keeps the incident angle and reflected angle equal. The sensor has an angle scanning accuracy of 0.002°, response sensitivity of 3.72 × 10−6 RIU (refractive index unit), and an angle scanning range of 30°–74°. The overall size of the system is only 480 mm × 150 mm × 180 mm. The portable SPR sensor was used to detect nucleic acid hybridization on a gold film chip modified with bovine serum albumin (BSA). The result revealed that the sensor had high sensitivity and fast response, and could successfully accomplish the hybridization detection of target DNA solution of 0.01 μmol/mL.

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

AbstractThe pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD), which is a major problem in East Asia and West Europe. Quick identification of PWN is needed to prevent the dispersal of PWD to healthy forests. Various detection methods of PWN have been developed using anatomical characters and molecular markers. These methods are not suitable for rapid diagnosis because it is difficult to distinguish B. xylophilus from the non‐pathogenic species Bursaphelenchus mucronatus based on morphological characters without expertise in nematode taxonomy and most PCR or isothermal amplification detection methods require time‐consuming processes. In this study, we developed an on‐site PWN detection method using a recombinase polymerase amplification (RPA) assay with a novel extraction buffer (DAP buffer). This new PWN detection method is able to extract genomic DNA from PWN in pinewood by simple buffer consisting of sodium hydrate, polyethylene glycol 200 and dimethyl sulfoxide in 10 min without using the experimental devices and able to distinguish between B. xylophilus and other Bursaphelenchus spp. by amplifying the species‐specific 5S rDNA fragment of B. xylophilus in 10 min. Taken together, our protocol can obtain the result for the detection of PWN in pine tree samples within 30 min. This result suggests that RPA/DAP assay is much faster, easier and cheaper than the conventional methods for detecting PWN.

粘液胞子虫性やせ病原因虫の検出に用いるLAMP法の開発

本研究では，粘液胞子虫性やせ病原因虫Enteromyxum leeiおよびSphaerospora fuguを検出するためのLAMP法を開発した。LAMP法のプライマーは，small subunit ribosomal RNA遺伝子領域を標的として設計した。LAMP法の最適な反応時間および反応温度を検討した結果，各原因虫ともに62°Cで60分間の反応を行うことで検出できることが示された。また，他の粘液胞子虫から抽出したDNAとの交差反応は見られず，反応特異性が認められた。さらに，当該LAMP法は，同じ遺伝子を標的としたPCR法よりも100～1,000倍の検出感度を示した。本研究で開発したE. leeiおよびS. fuguのLAMP法による検出系は，本疾病の検出・診断法として実用可能である。

A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes

Electrochemiluminescence (ECL) has been widely rendered for nucleic acid testing. Here, we integrate loop-mediated isothermal amplification (LAMP) with ECL technique for DNA detection and quantification. The target LAMP DNA bound electrostatically with [Ru(bpy)3]+2 on the carbon electrode surface, and an ECL reaction was triggered by tripropylamine (TPrA) to yield luminescence. We illustrated this method as a new and highly sensitive strategy for the detection of sequence-specific DNA from different meat species at picogram levels. The proposed strategy renders the signal amplification capacities of TPrA and combines LAMP with inherently high sensitivity of the ECL technique, to facilitate the detection of low quantities of DNA. By leveraging this technique, target DNA of Sus scrofa (pork) meat was detected as low as 1pg/µL (3.43×10−1copies/µL). In addition, the proposed technique was applied for detection of Bacillus subtilis DNA samples and detection limit of 10pg/µL (2.2×103copies/µL) was achieved. The advantages of being isothermal, sensitive and robust with ability for multiplex detection of bio-analytes makes this method a facile and appealing sensing modality in hand-held devices to be used at the point-of-care (POC).

Artificial Synthesis of Conserved Segment S Gene Fragment of Rift Valley Fever Virus and Preliminary Study of Its Reverse Transcription Loop-Mediated Isothermal Amplification Detection Method

Purpose: To develop a rapid detection method for Rift Valley fever virus (RVFV) diagnosis.Methods: According to the reference sequences of RVFV published in GenBank, nine overlapping polymerase chain reaction (PCR) primers and four specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) primers were designed using DNAStar and LAMP primer design software, respectively. Based on the synthesis of a conserved part of the RVFV S segment gene sequence using overlapping PCR, RT-LAMP assay was first established and evaluated after a series of tests, including, optimization of reaction conditions, and sensitivity and specificity tests.Result: A target RVFV S segment gene fragment of 288 bp was synthesised. The optimal reaction conditions for RT-LAMP assay were 63 °C for 45 min: the assay has a specific ladder-like pattern of amplification bands from about 120 bp. The lowest target gene copy number of RT-LAMP for RVFV detection was 70 copies. The assay showed good specificity as only the synthesised target RVFV gene was amplified with no amplification for the detection of Peste des petits ruminants virus, Epidemic encephalitis B virus, E. coli, Pasteurella multocida, or Salmonella.Conclusion: This study provides a rapid, sensitive, specific RT-LAMP method for RVFV detection.Keywords: Rift valley fever virus, Overlapping polymerase chain reaction, Reverse transcription loopmediated isothermal amplification, Rapid diagnosis test

Rapid and enzyme-free nucleic acid detection based on exponential hairpin assembly in complex biological fluids.

Herein, we have developed a rapid and enzyme-free nucleic acid amplification detection method that combined the exponential self-assembly of four DNA hairpins and the FRET pair Cy3 and Cy5. This strategy was very ingenious and rapid, and could detect nucleic acids at concentrations as low as 10 pM in 15 min in biological fluids.

Development of a Rapid Event-Specific Loop-Mediated Isothermal Amplification Detection Method for Genetically Modified Maize NK603

In the present study, a loop-mediated isothermal amplification (LAMP) system was developed for detecting NK603 maize, and flanking sequence in NK603 maize was used as the target template to design event specific LAMP primers. Different kinds of genetically modified (GM) or non GM crops were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer set for NK603 maize had good levels of specificity. The limit of detection (LOD) of the LAMP system was about 50 copies of the maize haploid genome. Moreover, the amplified reaction could be accomplished in about 1 h, and the LAMP amplification products tend to be far longer than conventional PCR products and thus easier to be visible to naked eyes via end point analysis. Therefore, the LAMP method developed by this study is visual, faster, and more sensitive, and does not need special equipment compared to traditional PCR technique, which is very useful for detecting transgenic maize aimed at screening the growing transgenic crops in the fields and detecting GM ingredients in imported and domestic foods.