Real-Time Recombinase Polymerase Amplification (RPA) Detection of Pseudomonas aeruginosa Using Magnetic Nano-Beads for DNA Extraction

2021 ◽  
Vol 13 (9) ◽  
pp. 1657-1665
Author(s):  
Yun Xu ◽  
Yanqi Wu ◽  
Ling Liang ◽  
Pingping Huang ◽  
Rui Chen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nosocomial Infection ◽  
Real Time ◽  
Dna Extraction ◽  
Pathogenic Bacteria ◽  
Magnetic Beads ◽  
Clinical Sample ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Time Saving

Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous nonfermentative gram-negative bacillus, residing in nature widely as a conditional pathogen that is commonly isolated from nosocomial infection cases, having a larger genome (5.5–7 Mbp). P. aeruginosa possesses great environmental adaptability and higher mutation rates, which accounts for its ability to resist antibiotics. Furthermore, multi-antibiotics-resistant P. aeruginosa has recently been established to be responsible for increased nosocomial infection incidence. Therefore, to detect, diagnose, and treat this life-threatening infection early enough, we introduced a real-time recombinase polymerase amplification (RPA) method of rapid P. aeruginosa detection, which is time-saving, convenient, highly sensitive, and more specific than the traditional bacterial culture method. System effectiveness and efficiency were then determined by a series of experiments, where specific RPA primers and probes accurately distinguished P. aeruginosa from other common pathogenic bacteria with optimal RPA reaction procedures set at 39 °C for 20 min. Additionally, specific primers and probes were designed based on the oprF gene sequence of P. aeruginosa. Subsequently, a real-time RPA system was used to analyze 20 cases of different clinical samples for P. aeruginosa detection in blood, open wound swab, and sputum. Results indicated that this system was suitable for bacterial analysis of common clinical sample types. The DNA extraction method used in this study was based on the magnetic beads method. In conclusion, this study established that the real-time RPA system was a rapid, specific, and sensitive method for clinical P. aeruginosa detection, which can be integrated into automatic high-throughput detection machines.

scholarly journals Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246573
Author(s):  
Sandeep K. Gupta ◽  
Qing Deng ◽  
Tanushree B. Gupta ◽  
Paul Maclean ◽  
Joerg Jores ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Clinical Samples ◽  
The Real ◽  
Positive Rate ◽  
Mycoplasma Ovipneumoniae

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

Are Three Items Sufficient to Measure Sense of Coherence?

2018 ◽  
Vol 34 (4) ◽  
pp. 229-237 ◽  
Author(s):  
Francesca Chiesi ◽  
Andrea Bonacchi ◽  
Caterina Primi ◽  
Alessandro Toccafondi ◽  
Guido Miccinesi
Keyword(s):  
Sense Of Coherence ◽  
Convergent Validity ◽  
Clinical Sample ◽  
Clinical Samples ◽  
Depression Symptoms ◽  
Time Saving ◽  
Positive Role ◽  
Patients With Cancer ◽  
Measure Sense

Abstract. The present study aimed at evaluating if the three-item sense of coherence (SOC) scale developed by Lundberg and Nystrom Peck (1995) can be effectively used for research purpose in both nonclinical and clinical samples. To provide evidence that it represents adequately the measured construct we tested its validity in a nonclinical (N = 658) and clinical sample (N = 764 patients with cancer). Results obtained in the nonclinical sample attested a positive relation of SOC – as measured by the three-item SOC scale – with Antonovsky’s 13-item and 29-item SOC scales (convergent validity), and with dispositional optimism, sense of mastery, anxiety, and depression symptoms (concurrent validity). Results obtained in the clinical sample confirmed the criterion validity of the scale attesting the positive role of SOC – as measured by the three-item SOC scale – on the person’s capacity to respond to illness and treatment. The current study provides evidence that the three-item SOC scale is a valid, low-loading, and time-saving instrument for research purposes on large sample.

scholarly journals ANTIBACTERIAL RESISTANCE PATTERN OF PSEUDOMONAS AERUGINOSA ISOLATED FROM CLINICAL SAMPLES AT A GENERAL HOSPITAL IN PADANG, WEST SUMATRA, INDONESIA

2017 ◽  
Vol 10 (8) ◽  
pp. 158
Author(s):  
Rustini Rustini ◽  
Jamsari Jamsari ◽  
Marlina Marlina ◽  
Nasrul Zubir ◽  
Yori Yuliandra
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Clinical Sample ◽  
Opportunistic Pathogen ◽  
Positive Control ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Resistance Pattern ◽  
Tract Infections ◽  
Almost All

Objectives: Pseudomonas aeruginosa is an opportunistic pathogen that has an innate resistance to some antibiotics. This bacterium is one of the mostcommon causes of nosocomial infections that include surgical wound infections, burns, and urinary tract infections. The bacteria have been reportedlyresistant to many antibiotics and have developed multidrug resistance (MDR). The objective of the study was to determine the resistance pattern ofP. aeruginosa isolated from clinical samples of patients against some major antibiotics.Methods: Isolates of P. aeruginosa were obtained from clinical sample of urine, sputum, swabs, pus, feces, and blood and cultured in cetrimide agar. P.aeruginosa ATCC 27853 was used as a positive control. The antibacterial susceptibility testing was conducted against 13 antibiotics: Ceftazidime, cefotaxime,ceftriaxone, cefoperazone, ciprofloxacin, levofloxacin, ofloxacin, gentamicin, amikacin, piperacillin, ticarcillin, meropenem, and imipenem. The examinationwas carried out using agar diffusion method of Kirby-Bauer and following the standards from Clinical and Laboratory Standards Institute (CLSI).Results: The results showed that bacterial resistance was established against all tested antibiotics. The highest number of resistance was shownagainst ceftriaxone (44.21%), whereas the most susceptibility was exhibited against amikacin (only 9.47% of resistance). MDR P. aeruginosa (MDRPA)was detected on almost all clinical samples tested, except the feces. The sample with the highest percentage of MDRPA was the pus.Conclusion: The study concludes that the most effective antibiotic against P. aeruginosa is amikacin (91.51%), whereas the most resistance is exhibited to ceftriaxone (43.16%).

scholarly journals Molecular characterization of resistance mechanisms in Pseudomonas aeruginosa isolates resistant to carbapenems

2018 ◽  
Vol 11 (12) ◽  
pp. 935-943 ◽  
Author(s):  
Mona Shaaban ◽  
Ahmed Al-Qahtani ◽  
Mohammed Al-Ahdal ◽  
Rasha Barwa
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Real Time ◽  
Real Time Pcr ◽  
Treatment Options ◽  
Carbapenem Resistance ◽  
Pulse Field ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Carbapenem Resistant

Introduction: Emergence of carbapenem resistance in Pseudomonas aeruginosa increases the therapeutic dilemma. In this study, we investigated various mechanisms involved in the resistance of P. aeruginosa clinical isolates to carbapenems. Methodology: P. aeruginosa isolates were isolated from different clinical samples. The antimicrobial susceptibility was evaluated by disc diffusion method. Carbapenemases were detected among carbapenem resistant isolates. Expression level of mexB and oprD was determined by real-time PCR. Molecular relatedness among isolates was detected based on pulse-field gel electrophoresis (PFGE). Results: Ninety P. aeruginosa isolates were purified from clinical specimens. High levels of resistance to imipenem and meropenem were detected in 16 isolates. PCR analysis of carbapenemases indicated the prevalence of Verona integron-encoded metallo-beta-lactamase (VIM); four isolates produced only VIM enzymes (VIM-1 or VIM-2), while the remaining twelve co-produced both VIM-1 or VIM-2 and NDM enzymes. Additionally, real-time PCR analysis elucidated high expression levels of mexB in seven of the carbapenem resistant isolates and low expression of oprD in seven isolates. The identified carbapenem-resistant isolates were clustered into eleven PFGE profiles where clusters E1 and E2 involved isolates exhibiting multiple carbapenemase genes (blaNDM-1, blaVIM-1 and blaVIM-2). Conclusion: Various mechanisms underlying carbapenem resistance have been detected in our P. aeruginosa cohort of isolates. Emergence of P. aeruginosa as a reservoir of multiple carbapenemases is increasing over time limiting the treatment options to this serious infection. This increases the urgency for infection control practices to reduce the incidence of this infection.

scholarly journals 261.Probiotic Lactobacillus in seminal plasma

2004 ◽  
Vol 16 (9) ◽  
pp. 261 ◽  
Author(s):  
G. Sivaramakrishnan ◽  
M. J. Jasper ◽  
S. O'Leary ◽  
S. A. Robertson
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Real Time ◽  
Seminal Plasma ◽  
Pathogenic Bacteria ◽  
Reproductive Tract ◽  
Bacterial Species ◽  
Female Reproductive Tract ◽  
Plasma Samples ◽  
Culture Techniques

Commensal bacteria of the Lactobacillus genus are implicated in beneficial 'probiotic' roles in the gut and other mucosal tissues. Their presence reduces the incidence of pathogenic infections, both passively and via production of antimicrobial substances, and through Toll-like receptor-mediated activation of cytokine expression in host tissues. Lactobacilli are present in the female reproductive tract but have not been examined in the male. This study aimed to investigate, by selective culture techniques and real-time quantitative PCR, the prevalence in boar seminal plasma of Lactobacilli compared with other pathogenic bacteria. Using acidified Rogosa Agar, Lactobacilli were cultured from 3/3 fresh semen samples and were found to be most prevalent in the first fraction of the ejaculate. For PCR, DNA was extracted from reference bacterial cultures and archived seminal plasma samples from 40 healthy boars. Bacterial species-specific primers targeting Lactobacillus 16s and 16s-23s rDNA sequences, and Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus-specific Sau3AI, oprL, and 16s rDNA genes respectively, were used in real-time PCR assays employing SYBRgreen (Applied Biosystems) technology. Lactobacilli were detected in 22/40 (55%) of seminal plasma samples, while pathogenic bacteria were detected in <10% of samples (Staphylococcus aureus, 1/40; Pseudomonas aeruginosa, 2/40; and Bacillus, 3/40). The Lactobacillus content of individual boars ranged from 1.5 to 15 × 106 cells/mL, and within boars, content varied within 30% of the mean value in successive samples over a 6-month period. We conclude that Lactobacilli are present in abundance in boar seminal plasma compared to other potentially pathogenic bacteria. These bacteria may protect the male tract from pathogen infection, and after ejaculation, may influence the female immune response to male antigens. Ongoing studies will investigate whether Lactobacilli abundance in seminal plasma correlates with boar fertility, and examine the potential value of improving reproductive performance in pigs and other species by administration of probiotic agents.

Evaluation of DNA extraction methods and dilution treatment for detection and quantification of Acanthamoeba in water and biofilm by real-time PCR

2010 ◽  
Vol 62 (9) ◽  
pp. 2141-2149 ◽  
Author(s):  
Ching-Wen Chang ◽  
Ying-Chieh Wu
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Acanthamoeba Castellanii ◽  
Extraction Methods ◽  
Cell Number ◽  
Pcr Analysis ◽  
Human Pathogens ◽  
Dna Extraction Methods

Acanthamoeba, human pathogens and natural hosts of pathogenic bacteria, may be accurately detected and quantified by real-time PCR if Acanthamoeba DNA are properly extracted and PCR inhibitors are effectively eliminated. However, the optimization of DNA extraction methods has not been reported for Acanthamoeba. This study compared the effectiveness of two DNA extraction/purification methods (FastDNA® Spin Kit for soil and Wizard® SV genomic DNA Purification System) by using trophozoites and cysts of Acanthamoeba castellanii and water and biofilm samples of cooling towers. DNA of A. castellanii extracted with the FastDNA® Kit and quantified by TaqMan PCR resulted in a lower variation (CV of Ct &lt; 3%), greater linearity (R2=0.99), and higher slopes (1.177–1.187 log fg DNA/log cell number) as compared to that by the Wizard® Kit. For field testing, the number of Acanthamoeba-positive samples and the Acanthamoeba DNA quantity were both greater with the FastDNA® Kit than with the Wizard® Kit (P=0.016 and &lt;0.0001, respectively). Beneficial effects with dilutions of extracted DNA were also revealed with the FastDNA® Kit (P=0.0003). In conclusion, DNA extraction by the FastDNA® Kit coupled with dilution of extracted DNA and PCR analysis are recommended for detecting and quantifying environmental Acanthamoeba.

scholarly journals A Rapid and Sensitive Detection Method for Pseudomonas aeruginosa Using Visualized Recombinase Polymerase Amplification and Lateral Flow Strip Technology

2021 ◽  
Vol 11 ◽  
Author(s):  
Haitao Yang ◽  
Yan Wang ◽  
Qiankun Yang ◽  
Hui Fan ◽  
Lei Wang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Detection Method ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Coincidence Rate ◽  
Kappa Index ◽  
Biochemical Method ◽  
Lateral Flow Strip ◽  
Index Value

Pseudomonas aeruginosa is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the genetic disorder cystic fibrosis. Early diagnosis in infected patients and monitoring P. aeruginosa contamination is therefore of great importance in controlling disease spread and development with timely drugs intervention treatment and cut off infection source. Traditional culture-biochemical methods are time consuming and highly dependent on technicians and expensive instruments. To address these challenges, the present study aimed to develop a rapid, sensitive, and specific, on-site detection method for P. aeruginosa based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology. The experimental process included screening and modification of primer and probe sets targeting the unique virulence gene elastase B (lasB); specificity detection in 29 strains of P. aeruginosa and 23 closely-related pathogenic bacteria; sensitivity measurements with gradient-diluted P. aeruginosa genomic DNA and probit regression analysis; and clinical application evaluation using 574 patients samples and calculating coincidence rate and kappa index value in comparison with the culture-biochemical method. The P. aeruginosa RPA-LFS assay could complete the amplification process at 37°C constant temperature within 30 min and results could be visualized by the naked eye within 10 min on LFS. The assay displayed high sensitivity with a limit of detection of 3.05 CFU/reaction. It also demonstrated high specificity by showing no cross reaction with other pathogenic bacteria, and rapidness by being completed in less than an hour. Furthermore, when used with clinical samples, the assay had a coincidence rate of 98.26% with the culture-biochemical method and a kappa index value of 0.9433. These data indicate that the RPA-LFS assay represents a major improvement for P. aeruginosa detection, especially in resource-limited areas.

scholarly journals Development and Evaluation of Real-Time Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid and Sensitive Detection of West Nile Virus in Human Clinical Samples

2021 ◽  
Vol 10 ◽  
Author(s):  
Priyanka Singh Tomar ◽  
Sanjay Kumar ◽  
Sapan Patel ◽  
Jyoti S. Kumar
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Reverse Transcription ◽  
Febrile Illness ◽  
Posterior Uveitis ◽  
Qpcr Assay ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
West Nile Fever ◽  
West Nile

West Nile virus (WNV) causes West Nile fever and encephalitis worldwide. Currently, there are no effective drugs or vaccines available in the market to treat WNV infection in humans. Hence, it is of paramount importance to detect WNV early for the success of the disease control programs and timely clinical management in endemic areas. In the present paper, we report the development of real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for rapid and real-time detection of WNV targeting the envelope (env) gene of the virus. The RPA reaction was performed successfully at 39°C for 15 min in a real-time thermal cycler. The sensitivity of this assay was found similar to that of the quantitative real-time RT PCR (RT-qPCR) assay, which could detect 10 copies of the gene. The efficacy of the assay was evaluated with a panel of 110 WN suspected human samples showing the signs of retinitis, febrile illness and acute posterior uveitis. In comparison with RT-qPCR, RT-RPA showed a specificity of 100% (CI, 95.07–100%) and sensitivity of 96.15% (CI, 80.36–99.90%) with a negative (NPV) and positive predictive value (PPV) of 98.65 and 100%, respectively. The level of agreement between RT-RPA and reference RT-qPCR assay was shown to be very high. The turnaround time of real-time RPA assay is about 10-20 times faster than the RT-qPCR, which confirms its utility in the rapid and sensitive diagnosis of WNV infection. To the best of our knowledge, this is the first report which deals with the development of real-time RT-RPA assay for simple, rapid, sensitive, and specific detection of WNV in human clinical samples. The present RT-RPA assay proves to be a powerful tool that can be used for the rapid diagnosis of a large number of patient samples in endemic settings.

scholarly journals Development of a Recombinase Polymerase Amplification Fluorescence Assay for the Detection of Canine Adenovirus 2

2021 ◽  
Vol 8 ◽  
Author(s):  
Li Xiao ◽  
Mengdi Zhang ◽  
Zhige Tian ◽  
Ye Ge ◽  
Tongyuan Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Canine Distemper Virus ◽  
Pathogen Detection ◽  
Detection Method ◽  
Adenovirus Type ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Clinical Applicability ◽  
Field Samples

Canine adenovirus type 2 (CAdV-2) is often found in co-infections with other pathogens causing canine infectious respiratory disease (CIRD). Rapid, efficient, and convenient pathogen detection is the best approach for early confirmatory diagnosis. In this study, we developed and evaluated a rapid real-time recombinase polymerase amplification (RPA) assay for detection of canine adenovirus 2 (CAV), which can detect CAV within 15 min at 39°C. The detection limit that assay was 214 copies/μl DNA molecules per reaction. The specificity was indicated by a lack of cross-reaction with canine distemper virus (CDV), canine coronavirus (CCV), and canine parvovirus (CPV). Field and clinical applicability of this assay were evaluated using 86 field samples. The coincidence rate of the detection results for clinical samples between CAV-RPA and qPCR was 97.7%. In summary, the real-time CAV-RPA analysis provides an efficient, rapid and sensitive detection method for CAV.

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