Detection of Mycoplasma Contamination of Cell Culture by A Loop-Mediated Isothermal Amplification Method.
ABSTRACT: Objective:Mycoplasmas are major contaminants of cell culture and affect in vitro biological and diagnostic tests. Mycoplasma detection is conducted using culture and molecular methods. These methods vary in terms of accuracy, reliably and sensitivity. Loop-mediated isothermal amplification (LAMP) is used to amplify target DNA in a highly specific and rapid manner. This study aimed to develop a LAMP method for rapid detection of Mycoplasma in culture samples. Materials and Methods:In this descriptive laboratory study, for LAMP detection of Mycoplasma contaminations in cell culture, we used primers specifically designed for targeting the 16S rRNA conserved gene of Mycoplasma spp. For a positive control structure, 16S rRNA amplified based on PCR, was cloned in a plasmid vector and sequenced. The assay specificity was evaluated using Mycoplasma genomic DNA and a panel containing genomes of gram-positive and gram-negative organisms. Results:In this study, the method developed for detection of Mycoplasma contamination of cell cultures was a rapid, sensitive and cost-effective LAMP approach. The results demonstrated that this method benefits from high specificity (100%) for amplification of Mycoplasma strains and high speed (multiplication within 60 minutes), while it does not require expensive laboratory equipment compared to those needed for polymerase chain reaction (PCR)-based detection. Conclusion:Our study is the first report about application of LAMP assay based on 16S rRNA gene for detection of Mycoplasma strains; this technique could be considered a useful tool for rapid detection of contamination of cell culture.
Project description:We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.
Project description:Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies.
Project description:Loop-mediated isothermal amplification (LAMP) is a nucleic acid method which has been used to identify mycobacteria including Mycobacterium tuberculosis in clinical microbiology laboratory and point of care settings. Previously published LAMP protocols for detection of mycobacterial species used conventional specific primer and targeted the 16S rRNA, gyrB, and insertion sequence genes. We developed and evaluated a LAMP assay targeting a mycobacterial rpoB gene conserved sequence and incorporating degenerate primers. This assay allowed consensus detection of mycobacterial species from pure culture, clinical respiratory tract samples, and mycobacteria growth indicator tube (MGIT) liquid-based culture medium. A panel of twenty mycobacterial species were successfully detected at detection thresholds of 102 CFU/mL and 103 CFU/mL when respectively performed on pure culture suspension or sputum and MGIT broth. The inclusion of degenerate bases in LAMP primers increased the diversity of mycobacterial species identified by the assay without negatively affecting analytical sensitivity. LAMP-based consensus detection of multiple pathogens can be achieved with degenerate primers therefore allowing the design of rapid multi-disease screening assays. Despite high analytical sensitivity, species specificity and the advantageous operational characteristics of LAMP over PCR, challenges such as potential ambiguity in visual interpretation of results and occasional non-specific amplification precludes the implementation of novel LAMP assay in routine diagnostics both in centralized and point-of-care laboratory.
Project description:A loop-mediated isothermal amplification (LAMP) system, the illumigene Mycoplasma DNA amplification assay (Meridian Bioscience, Inc., Cincinnati, OH) was evaluated to determine its analytical sensitivity, specificity, and clinical application in comparison to historic culture in a collection of archived respiratory specimens. The illumigene limit of detection was ?88 CFU/reaction for 10 Mycoplasma pneumoniae reference strains. This assay correctly identified 36 M. pneumoniae reference strains and clinical isolates from various geographic origins, including both of the main subtypes. No cross-reactions were detected with other mycoplasmas, ureaplasmas, other bacterial species, viruses, yeasts, or human DNA. Among 214 respiratory specimens previously cultured for M. pneumoniae, when real-time PCR with bidirectional sequencing of the PCR products was used to resolve discrepancies, the sensitivity was 22 of 22 (100%) and the specificity was 190 of 192 (99%). This commercial LAMP assay is a useful rapid method for detecting M. pneumoniae in clinical specimens. Additional prospective clinical trials with direct comparison to culture and PCR are warranted.
Project description:Background:Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) worldwide, especially among children and debilitated populations. The present study aimed to investigate a loop-mediated isothermal amplification (LAMP) technique for rapid detection of M. pneumoniae in clinical specimens collected from patients with pneumonia. Methods:Throat swabs were collected from 110 outpatients who suffered from pneumonia. Throat swab samples were obtained from patients referred to the hospital outpatient clinics of Tehran University hospitals, Iran in 2017. The presence of M. pneumoniae in the clinical specimens was evaluated by LAMP, PCR and culture methods. Sensitivity and specificity of the LAMP and PCR assays were also determined. Results:Out of 110 specimens, LAMP assay detected M. pneumoniae in 35 specimens. Detection limit of the LAMP assay was determined to be 33fg /?L or ? 40 genome copies/reaction. Moreover, no cross-reaction with genomic DNA from other bacteria was observed. Only 25 specimens were positive by the culture method. The congruence between LAMP assay and culture method was 'substantial' (?=0.77). Specificity and sensitivity of LAMP assay were 88.2%, 100% in compare with culture. However, the congruence between LAMP assay and PCR assay was 'almost perfect' (?=0.86). Specificity and sensitivity of LAMP assay were 92.5%, 100% in compare with PCR. Conclusion:Overall, the LAMP assay is a rapid and cost-efficient laboratory test in comparison to other methods including PCR and culture. Therefore, the LAMP method can be applied in identification of M. pneumoniae isolates in respiratory specimens.
Project description:Introduction:Mycoplasma gallisepticum is considered the most pathogenic and economically significant avian Mycoplasma spp. for the worldwide poultry industry. The aim of this study was to develop a novel and sensitive real-time loop-mediated isothermal amplification (LAMP) assay based on the amplification of its mgc2 gene sequence for its rapid molecular detection in poultry. Material and Methods:Blood samples from 300 broiler and layer chickens were screened using a rapid serum agglutination (RSA) test. A real-time LAMP reaction was conducted with seropositive swab samples at 60ºC for 90 min in an ESEQuant tube scanner using 6-carboxyfluorescein as the reporting dye. Results:The sensitivity of the developed assay was 10 fg/µL of DNA. The assay was found 100% specific, showing no cross-reactivity with other avian Mycoplasma species. The proportion found of the positive samples by the real-time LAMP was 58%. In comparison, the RSA was found to detect 52% of positive cases. Conclusion:The mgc2 real-time LAMP emerged as a more sensitive and accurate method for molecular detection of M. gallisepticum than RSA. Robustness and precision give it applicability as a potential field diagnostic tool for M. gallisepticum control. The study will be beneficial in reducing economic losses that M. gallisepticum inflicts on the poultry industry. This is the first reported development of a real-time LAMP assay based on the amplification of the mgc2 gene sequence using an ESEQuant tube scanner for galline M. gallisepticum detection.
Project description:We developed a new loop-mediated isothermal amplification (LAMP) method to detect rrs, a 16S rRNA gene of pathogenic Leptospira spp. in urine. The method enables detection of two leptospiral cells per reaction mixture following boiling of urine specimens. The sensitivity of this method is higher than that of culture or of flaB nested PCR.
Project description:BACKGROUND: Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of Vibrio parahaemolyticus. RESULTS: The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 x 10(2) CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13-22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination. CONCLUSION: The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V. parahaemolyticus in seafood.
Project description:Contagious bovine mastitis caused by <i>Mycoplasma bovis</i> and other <i>Mycoplasma</i> species including <i>Mycoplasma californicum</i>, <i>Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma arginini</i>, and <i>Mycoplasma canadense</i> is an economical obstacle affecting many dairy herds throughout California and elsewhere. Routine bacteriological culture-based assays for the pathogens are slow and subject to false-positive results due to the presence of the related, non-pathogenic species <i>Acholeplasma laidlawii</i>. To address the need for rapid and accurate detection methods, a new TaqMan multiplex, quantitative real-time PCR (qPCR) assay was developed that targets the 16S rRNA gene of <i>Mycoplasma, rpoB</i> gene of <i>M. bovis</i>, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of <i>A. laidlawii</i>. qPCR amplification efficiency and range of detection were similar for individual assays in multiplex as when performed separately. The multiplex assay was able to distinguish between <i>M. bovis</i> and <i>A. laidlawii</i> as well as detect <i>Mycoplasma</i> spp. collectively, including <i>Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini</i> and <i>Mycoplasma alkalescens</i>. In milk, the lower limit of detection of <i>M. bovis, M. californicum</i>, and <i>A. laidlawii</i> with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both <i>M. bovis</i> and <i>A. laidlawii</i> in milk when present in moderate (10<sup>3</sup> to 10<sup>4</sup> CFU/mL) to high (10<sup>6</sup> to 10<sup>7</sup> CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI [86.8-100]; <i>n</i> = 26) and diagnostic sensitivity (Se) was 92.3% (95% CI [74.9-99.1]; <i>n</i> = 26) for <i>Mycoplasma</i> species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI [90.5-100]; <i>n</i> = 37) and Se was 93.3% (95% CI [68.1-99.8]; <i>n</i> = 15) for <i>M. bovis</i>. Our assay can detect and distinguish among <i>M. bovis</i>, other prevalent <i>Mycoplasma</i> spp., and non-pathogenic <i>Acholeplasma laidlawii</i> for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.
Project description:<h4>Purpose</h4>Pseudomonas aeruginosa is the most common bacteria causing endophthalmitis after cataract surgery. Vitreous fluid culture and molecular studies are commonly used in clinical diagnoses, but have disadvantages, such as a long culture cycle and low detection sensitivity. Here, we report a loop-mediated isothermal amplification (LAMP) method combined with the nanoparticles-lateral flow biosensor (LFB) method for rapid and specific detection of P. aeruginosa.<h4>Methods</h4>A set of six primers was designed to target the OprL gene of P. aeruginosa. Genomic DNA extracted from several gram-negative and gram-positive bacteria was used to determine the sensitivity and specificity of the analysis. LAMP reactions were conducted at 65 °C for 50 minutes, and results were reported using the LFB method.<h4>Results</h4>The DNA template of P. aeruginosa was specifically recognized by the P. aeruginosa-LAMP-LFB (PA-LAMP-LFB) method as no cross reactions were observed for non-P. aeruginosa templates. The analytical sensitivity of our assay was 100 fg per test for the pure cultured DNA template, and the result obtained using the LFB was consistent with that of colorimetric indicator detection. The whole test could be completed within 1h. This method was used to detect P. aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae; only P. aeruginosa was positive. The positive rates of P. aeruginosa detected by a traditional culture method, the LAMP-LFB method, and the fluorescence quantitative polymerase chain reaction method were 17.7%, 17.7%, and 13.3%, respectively.<h4>Conclusions</h4>The P. aeruginosa-LAMP-LFB method established here is a rapid, specific, and sensitive method for the detection of P. aeruginosa, which can be widely used.