scholarly journals Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum

2020 ◽  
Vol 58 (5) ◽  
Author(s):  
Le Jiang ◽  
Philip Ching ◽  
Chien-Chung Chao ◽  
J. Stephen Dumler ◽  
Wei-Mei Ching
Keyword(s):  
Anaplasma Phagocytophilum ◽  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Limit Of Detection ◽  
Dna Amplification ◽  
Recombinase Polymerase Amplification ◽  
Detection Methods ◽  
Content Type ◽  
Amplification Assay ◽  
Nonspecific Amplification

ABSTRACT Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum. The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum. Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

scholarly journals Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae

2015 ◽  
Vol 53 (9) ◽  
pp. 2810-2815 ◽  
Author(s):  
Anne Liljander ◽  
Mingyan Yu ◽  
Elizabeth O'Brien ◽  
Martin Heller ◽  
Julia F. Nepper ◽  
...  
Keyword(s):  
Dna Amplification ◽  
Recombinase Polymerase Amplification ◽  
Target Sequence ◽  
Content Type ◽  
Mycoplasma Capricolum ◽  
Contagious Caprine Pleuropneumonia ◽  
Geographical Regions ◽  
Amplification Assay ◽  
Simulated Field ◽  
High Degree

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused byMycoplasma capricolumsubsp.capripneumoniaethat affects goats in Africa and Asia. Current available methods for the diagnosis ofMycoplasmainfection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection ofM. capricolumsubsp.capripneumoniae. We developed the assay using a specific target sequence inM. capricolumsubsp.capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 103and 5 × 104cells/ml were obtained using genomic DNA and bacterial culture fromM. capricolumsubsp.capripneumoniaestrain ILRI181, while no amplification was obtained from 71 relatedMycoplasmaisolates or from theAcholeplasmaor thePasteurellaisolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.

scholarly journals Disruption of VirB6 Paralogs in Anaplasma phagocytophilum Attenuates Its Growth

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Francy L. Crosby ◽  
Ulrike G. Munderloh ◽  
Curtis M. Nelson ◽  
Michael J. Herron ◽  
Anna M. Lundgren ◽  
...  
Keyword(s):  
Anaplasma Phagocytophilum ◽  
Pathogenic Bacteria ◽  
Tandem Repeats ◽  
Model Systems ◽  
Accession Number ◽  
Secretion Systems ◽  
Altered Expression ◽  
Content Type ◽  
Obligate Intracellular

ABSTRACT Many pathogenic bacteria translocate virulence factors into their eukaryotic hosts by means of type IV secretion systems (T4SS) spanning the inner and outer membranes. Genes encoding components of these systems have been identified within the order Rickettsiales based upon their sequence similarities to other prototypical systems. Anaplasma phagocytophilum strains are obligate intracellular, tick-borne bacteria that are members of this order. The organization of these components at the genomic level was determined in several Anaplasma phagocytophilum strains, showing overall conservation, with the exceptions of the virB2 and virB6 genes. The virB6 loci are characterized by the presence of four virB6 copies (virB6-1 through virB6-4) arranged in tandem within a gene cluster known as the sodB-virB operon. Interestingly, the virB6-4 gene varies significantly in length among different strains due to extensive tandem repeats at the 3′ end. To gain an understanding of how these enigmatic virB6 genes function in A. phagocytophilum, we investigated their expression in infected human and tick cells. Our results show that these genes are expressed by A. phagocytophilum replicating in both cell types and that VirB6-3 and VirB6-4 proteins are surface exposed. Analysis of an A. phagocytophilum mutant carrying the Himar1 transposon within the virB6-4 gene demonstrated that the insertion not only disrupted its expression but also exerted a polar effect on the sodB-virB operon. Moreover, the altered expression of genes within this operon was associated with the attenuated in vitro growth of A. phagocytophilum in human and tick cells, indicating the importance of these genes in the physiology of this obligate intracellular bacterium in such different environments. IMPORTANCE Knowledge of the T4SS is derived from model systems, such as Agrobacterium tumefaciens. The structure of the T4SS in Rickettsiales differs from the classical arrangement. These differences include missing and duplicated components with structural alterations. Particularly, two sequenced virB6-4 genes encode unusual C-terminal structural extensions resulting in proteins of 4,322 (GenBank accession number AGR79286.1) and 9,935 (GenBank accession number ANC34101.1>) amino acids. To understand how the T4SS is used in A. phagocytophilum, we describe the expression of the virB6 paralogs and explore their role as the bacteria replicate within its host cell. Conclusions about the importance of these paralogs for colonization of human and tick cells are supported by the deficient phenotype of an A. phagocytophilum mutant isolated from a sequence-defined transposon insertion library.

Real-Time Nucleic Acid–Based Detection Methods for Pathogenic Bacteria in Food

2004 ◽  
Vol 67 (4) ◽  
pp. 823-832 ◽  
Author(s):  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Food Microbiology ◽  
Detection Methods ◽  
Target Sequence ◽  
Bacterial Enumeration

Quality assurance in the food industry in recent years has involved the acceptance and implementation of a variety of nucleic acid–based methods for rapid and sensitive detection of food-associated pathogenic bacteria. Techniques such as polymerase chain reaction have greatly expedited the process of pathogen detection and have in some cases replaced traditional methods for bacterial enumeration in food. Conventional PCR, albeit sensitive and specific under optimized conditions, obligates the user to employ agarose gel electrophoresis as the means for endpoint analysis following sample processing. For the last few years, a variety of real-time PCR chemistries and detection instruments have appeared on the market, and many of these lend themselves to applications in food microbiology. These approaches afford a user the ability to amplify DNA or RNA, as well as detect and confirm target sequence identity in a closed-tube format with the use of a variety of fluorophores, labeled probes, or both, without the need to run gels. Such real-time chemistries also offer greater sensitivity than traditional gel visualization and can be semiquantitative and multiplexed depending on the specific experimental objectives. This review emphasizes the current systems available for real-time PCR–based pathogen detection, the basic mechanisms and requirements for each, and the prospects for development over the next few years in the food industry.

Different Methods for Molecular and Rapid Detection of Human Novel Coronavirus

2021 ◽  
Vol 27 ◽  
Author(s):  
Mohamad Hesam Shahrajabian ◽  
Wenli Sun ◽  
Qi Cheng
Keyword(s):  
Nucleic Acid ◽  
Rapid Detection ◽  
Pathogenic Bacteria ◽  
Scientific Information ◽  
Meat Products ◽  
Lamp Assay ◽  
Dna Amplification ◽  
Digital Pcr ◽  
Recombinase Polymerase Amplification ◽  
Nucleic Acid Amplification

Introduction: While PCR has been recognized as one of the appropriate ways to diagnosis of infectious diseases, Loop-mediated isothermal amplification (LAMP), which is a nucleic acid amplification method, can be considered as an alternative to PCR, and it is faster, cost-effective, and easier to perform than nested PCR. Patients and Methods: Keywords were searched in PubMed/MEDLINE, Scopus and Institute for Scientific Information Web of Science, as well as the search engine of Google Scholar. Keywords were PCR, LAMP, RAA, RPA, Virus and COVID-19. Results: LAMP technology has been extensively applied for the detection of human pathogenic bacteria, crop pests, pathogenic organisms and components in meat products. A new isotheral method, Recombinase polymerase amplification (RPA), can amplify the DNA as well as RPA. RPA has benefited from isothermal PCR and both simplicity and rapid amplification. Recombinase aided amplification (RAA) assay has been favorably used in the detection of bacterial and viral pathogens and solved the technical difficulties posed by DNA amplification methods because it does not need thermal denaturation of the template and employs at a low and constant temperature. Conclusions: Reverse transcription polymerase chain reaction, digital PCR, LAMP, nicking endonuclease amplification reaction, recombinase polymerase amplification, and clustered regularly interspaced short palindromic repeats are different nucleic acid amplification tests of COVID-19. LAMP methods can be more specific than qPCR and immunoassays. The LAMP assay can be applied for rapid detection of SARS-CoV, MERS-CoV, SARS-CoV-2, and influenza, because LAMP is a highly sensitive and specific DNA/RNA amplification technique.

scholarly journals Rapid and Sensitive Detection of Vibrio vulnificus Using CRISPR/Cas12a Combined With a Recombinase-Aided Amplification Assay

2021 ◽  
Vol 12 ◽  
Author(s):  
Xingxing Xiao ◽  
Ziqin Lin ◽  
Xianhui Huang ◽  
Jinfang Lu ◽  
Yan Zhou ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Detection Method ◽  
Limit Of Detection ◽  
Sensitive Detection ◽  
Detection Methods ◽  
Farmed Fish ◽  
Safety Control ◽  
Amplification Assay ◽  
Sophisticated Instrument ◽  
Aquatic Pathogen

Vibrio vulnificus is an important zoonotic and aquatic pathogen and can cause vibriosis in humans and aquatic animals (especially farmed fish and shrimp species). Rapid and sensitive detection methods for V. vulnificus are still required to diagnose human vibriosis early and reduce aquaculture losses. Herein, we developed a rapid and sensitive diagnostic method comprising a recombinase-aided amplification (RAA) assay and the CRISPR/Cas12a system (named RAA-CRISPR/Cas12a) to detect V. vulnificus. The RAA-CRISPR/Cas12a method allows rapid and sensitive detection of V. vulnificus in 40 min without a sophisticated instrument, and the limit of detection is two copies of V. vulnificus genomic DNA per reaction. Meanwhile, the method shows satisfactory specificity toward non-target bacteria and high accuracy in the spiked blood, stool, and shrimp samples. Therefore, our proposed rapid and sensitive V. vulnificus detection method, RAA-CRISPR/Cas12a, has great potential for early diagnosis of human vibriosis and on-site V. vulnificus detection in aquaculture and food safety control.

Immunomagnetic Separation in Microchannels: From MEMS to BioNEMS

2005 ◽  
Author(s):  
Ashok Sinha ◽  
Ranjan Ganguly ◽  
Ishwar K. Puri
Keyword(s):  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Contaminated Water ◽  
Selective Detection ◽  
Bacterial Detection ◽  
E Coli ◽  
Biological Environment ◽  
Innovative Concept

Conventional methods of monitoring and testing water quality involve collection of the sample to be tested and its subsequent analysis in a research laboratory for which some procedures may not be feasible or even accessible under certain field situations. Therefore, next generation sensors are required. Herein, an innovative concept that combines a micromixer and microparticle trap is proposed that should enable more rapid pathogen detection in contaminated water. In it, immunomagnetic separation (a procedure [1,2] that is well practiced in the field of immunochemistry) is scaled down from the benchtop to the microscale. Our design is generic, i.e., design is not limited to the detection of waterborne biological agents, but can be used for other forms of chemical analysis. Testing for waterborne bacteria requires analysis methods that must meet a number of challenging criteria. Time and sensitivity of analysis are the more important limitations. Bacterial detection methods have to be rapid and very sensitive since the presence of even a small pathogenic sample may sometimes constitute an infectious or otherwise harmful dose. Selective detection is also required because small numbers of pathogenic bacteria are often present in a complex biological environment along with many other nonpathogenic organisms. As an example, the infectious dosage of a pathogen such as E. coli O157:H7 or Salmonella is as low as 10 cells and the existing coliform standard for E. coli in water is 4 cells: 100 ml [3].

scholarly journals Recombinase Polymerase Amplification Assay for Field Detection of Tomato Bacterial Spot Pathogens

2019 ◽  
Vol 109 (4) ◽  
pp. 690-700 ◽  
Author(s):  
A. Strayer-Scherer ◽  
J. B. Jones ◽  
M. L. Paret
Keyword(s):  
Detection Limit ◽  
Pathogenic Bacteria ◽  
Diagnostic Methods ◽  
Recombinase Polymerase Amplification ◽  
Bacterial Spot ◽  
Field Detection ◽  
Field Samples ◽  
Amplification Assay ◽  
Polymerase Chain ◽  
Recombinase Polymerase Amplification Assay

Bacterial spot of tomato is caused by Xanthomonas gardneri, X. euvesicatoria, X. perforans, and X. vesicatoria. Current diagnostic methods for the pathogens are not in-field assays. Recombinase polymerase amplification (RPA) is ideal for in-field detection assays, because it is an isothermal technique that is rapid and more tolerant to inhibitors compared with polymerase chain reaction. Hence, novel RPA probes and primers were designed to amplify regions of the hrcN gene of X. gardneri, X. euvesicatoria, and X. perforans. The X. gardneri RPA is specific to X. gardneri with a detection limit of 106 CFU/ml and detected X. gardneri in lesions from naturally (n = 6) or artificially (n = 18) infected plants. The X. euvesicatoria RPA detects both X. euvesicatoria and X. perforans with a detection limit of 106 CFU/ml and detected both pathogens in plants artificially infected (n = 36) or naturally infected (n = 85) with either X. euvesicatoria or X. perforans. The X. perforans RPA is specific to X. perforans with a detection limit of 107 CFU/ml. Although the X. perforans RPA assay was unable to detect X. perforans from lesions, the X. euvesicatoria RPA was successfully used in field to detect X. perforans from symptomatic field samples (n = 31). The X. perforans RPA was then used to confirm the pathogen in the laboratory. The X. euvesicatoria and X. gardneri RPA is promising for rapid, real-time in-field detection of bacterial spot and one of the first developed among plant pathogenic bacteria.

scholarly journals Longitudinal Study of Shiga Toxin-ProducingEscherichia coliandCampylobacter jejunion Finnish Dairy Farms and in Raw Milk

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Anniina Jaakkonen ◽  
Hanna Castro ◽  
Saija Hallanvuo ◽  
Jukka Ranta ◽  
Mirko Rossi ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Dairy Farms ◽  
Raw Milk ◽  
Content Type ◽  
Fecal Shedding ◽  
Hygienic Measures ◽  
Cattle Herds ◽  
On Farm

ABSTRACTShiga toxin-producingEscherichia coli(STEC) andCampylobacter jejuniare notable health hazards associated with the consumption of raw milk. These bacteria may colonize the intestines of asymptomatic cattle and enter bulk tank milk via fecal contamination during milking. We studied the frequency of STEC O157:H7 andC. jejunicontamination in tank milk (n = 785) and the in-line milk filters of milking machines (n = 631) versus the frequency of isolation from cattle feces (n = 257) on three Finnish dairy farms for 1 year. Despite simultaneous isolation of STEC O157:H7 (17%) orC. jejuni(53%) from cattle, these bacteria were rarely isolated from milk filters (2% or <1%, respectively) and milk (0%). As revealed by phylogenomics, one STEC O157:H7 strain at a time was detected on each farm and persisted for ≤12 months despite rigorous hygienic measures.C. jejunistrains of a generalist sequence type (ST-883 and ST-1080) persisted in the herds for ≥11 months, and several otherC. jejunitypes were detected sporadically. Thestxgene carried by STEC was detected more frequently from milk filters (37%) than from milk (7%), suggesting that milk filters are more suitable sampling targets for monitoring than milk. A questionnaire of on-farm practices suggested lowerstxcontamination of milk when major cleansing in the barn, culling, or pasturing of dairy cows was applied, while a higher average outdoor temperature was associated with higherstxcontamination. Because pathogen contamination occurred despite good hygiene and because pathogen detection from milk and milk filters proved challenging, we recommend heat treatment for raw milk before consumption.IMPORTANCEThe increased popularity of raw milk consumption has created demand for relaxing legislation, despite the risk of contamination by pathogenic bacteria, notably STEC andC. jejuni. However, the epidemiology of these milk-borne pathogens on the herd level is still poorly understood, and data are lacking on the frequency of milk contamination on farms with cattle shedding these bacteria in their feces. This study suggests (i) that STEC contamination in milk can be reduced, but not prevented, by on-farm hygienic measures while fecal shedding is observable, (ii) that milk filters are more suitable sampling targets for monitoring than milk although pathogen detection from both sample matrices may be challenging, and (iii) that STEC andC. jejunigenotypes may persist in cattle herds for several months. The results can be utilized in developing and targeting pathogen monitoring and risk management on the farm level and contributed to the revision of Finnish legislation in 2017.

Simple and sensitive microbial pathogen detection using a label-free DNA amplification assay

2016 ◽  
Vol 52 (47) ◽  
pp. 7505-7508 ◽  
Author(s):  
Yuhuan Sun ◽  
Chuanqi Zhao ◽  
Zhengqing Yan ◽  
Jinsong Ren ◽  
Xiaogang Qu
Keyword(s):  
Magnetic Nanoparticles ◽  
Pathogen Detection ◽  
Dna Amplification ◽  
Label Free ◽  
Exonuclease Iii ◽  
Microbial Pathogen ◽  
Free Dna ◽  
Amplification Assay

A simple and facile strategy for sensitive pathogen detection has been developed by a combination of quaternized magnetic nanoparticles and a label-free exonuclease III-assisted DNA amplification assay.

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