Method For Detection Of Bacteria Research Articles

Rapid Quantitative Detection of Live Escherichia coli Based on Chronoamperometry.

The rapid quantitative detection of Escherichia coli (E. coli) is of great significance for evaluating water and food safety. At present, the conventional bacteria detection methods cannot meet the requirements of rapid detection in water environments. Herein, we report a method based on chronoamperometry to rapidly and quantitatively detect live E. coli. In this study, the current indicator i0 and the electricity indicator A were used to record the cumulative effect of bacteria on an unmodified glassy carbon electrode (GCE) surface during chronoamperometric detection. Through the analysis of influencing factors and morphological characterization, it was proved that the changes of the two set electrochemical indicator signals had a good correlation with the concentration of E. coli; detection time was less than 5 min, the detection range of E. coli was 104–108 CFU/mL, and the error range was <30%. The results of parallel experiments and spiking experiments showed that this method had good repeatability, stability, and sensitivity. Humic acid and dead cells did not affect the detection results. This study not only developed a rapid quantitative detection method for E. coli in the laboratory, but also realized a bacterial detection scheme based on the theory of bacterial dissolution and adsorption for the first time, providing a new direction and theoretical basis for the development of electrochemical biosensors in the future.

Implant surface culture may be a useful adjunct to standard tissue sampling culture for identification of pathogens accounting for fracture-device-related infection: a within-person randomized agreement study of 42 patients.

Background and purposeIdentification of pathogens causing fracture-device-related infection (FDRI) is always a challenge as the positive rate of standard tissue sampling culture (TSC) remains unsatisfactory. This study evaluates the efficiency of implant surface culture (ISC) as an adjunct to standard TSC for identification of FDRI-associated microorganisms.Patients and methodsBetween November 2020 and March 2022, patients diagnosed with FDRI defined by the International Fracture-Related Infection (FRI) Consensus Group, and indicated for implant removal, underwent both methods for bacteria detection. The test order of ISC and TSC was randomly selected for each patient included, as a withinperson randomized design. For ISC, the recovered implants were gently covered with tryptic soy agar after rinsing with normal saline twice, and then incubated at 37℃ 5% CO2 for up to 14 days. For TSC, 5 specimens were sampled and sent to the Clinical Laboratory of Southern Medical University Nanfang Hospital, Guangzhou, for culture and identification.Results42 consecutive patients were included, with a mean age of 46 years. The most frequent infection site and implant type were the tibia (21 cases) and plates with screws (30 cases), respectively. Altogether 21 patients were found with positive outcomes by both methods, and the identified pathogens were consistent. ISC found an additional 15 patients showing positive results, which were negative by TSC. Furthermore, the mean culture time of ISC was shorter than that of TSC (1.5 days vs. 3.2 days).InterpretationISC may be a useful adjunct to TSC for detection of bacteria causing FDRI, with a relatively higher positive rate and a shorter culture time.

Research progress on detection techniques for point-of-care testing of foodborne pathogens.

The global burden of foodborne disease is enormous and foodborne pathogens are the leading cause of human illnesses. The detection of foodborne pathogenic bacteria has become a research hotspot in recent years. Rapid detection methods based on immunoassay, molecular biology, microfluidic chip, metabolism, biosensor, and mass spectrometry have developed rapidly and become the main methods for the detection of foodborne pathogens. This study reviewed a variety of rapid detection methods in recent years. The research advances are introduced based on the above technical methods for the rapid detection of foodborne pathogenic bacteria. The study also discusses the limitations of existing methods and their advantages and future development direction, to form an overall understanding of the detection methods, and for point-of-care testing (POCT) applications to accurately and rapidly diagnose and control diseases.

Colorimetric nano-beacon and magnetic separation-based rapid and visual assay for gram-negative bacteria

Food-borne diseases caused by pathogenic bacteria are one of the serious factors affecting human health. However, the most commonly used detection methods for pathogenic bacteria not only require expensive instruments, but also take a long time due to the complicated and cumbersome detection process. Therefore, the development of a fast, simple, and low-cost detection method for pathogenic bacteria is crucial for food safety and human health. In this work, based on the high binding ability of antimicrobial peptide (AMP) and polymyxin B (PMB) to bacteria, combined with magnetic separation technology, a new enzyme-free colorimetric strategy was constructed to achieve visual detection of Gram-negative bacteria in complex samples. The sensor system was divided into the following two parts: a colorimetric signal amplification nanoprobe, which was modified with AMP to enable effective binding of the colorimetric probe to the surface of bacteria, and a PMB-modified magnetic nanobead (MNB), which was used as the capture and enrichment unit of Gram-negative bacteria, as a result of which PMB could effectively distinguish Gram-negative bacteria from Gram-positive bacteria. Under optimized conditions, the detection limit of the method for Gram-negative bacteria (e.g. E. coli (G−)) was as low as 10 CFU/mL, and it was successfully applied to complex real samples. In addition, the developed colorimetric sensor offered advantages, such as fast response, less time consumption, high sensitivity, and low cost. It can be expected to become a new diagnostic tool for on-site detection of pathogenic bacteria in remote areas.

Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach-Zehnder Interferometers Monolithically Integrated on Silicon Chips.

The consumption of water and milk contaminated with bacteria can lead to foodborne disease outbreaks. For this reason, the development of rapid and sensitive analytical methods for bacteria detection is of primary importance for public health protection. Here, a miniaturized immunosensor based on broadband Mach–Zehnder Interferometry for the simultaneous determination of S. typhimurium and E. coli O157:H7 in drinking water and milk is presented. For the assay, mixtures of bacteria solutions with anti-bacteria-specific antibodies were run over the chip, followed by solutions of biotinylated anti-species-specific antibody and streptavidin. The assay was fast (10 min for water, 15 min for milk), accurate, sensitive (LOD: 40 cfu/mL for S. typhimurium; 110 cfu/mL for E. coli) and reproducible. The analytical characteristics achieved combined with the small chip size make the proposed biosensor suitable for on-site bacteria determination in drinking water and milk samples.

A new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of marine-derived Bacillus velezensis B-9987

Marine-derived Bacillus velezensis B-9987 is an important biocontrol bacterium with a broad-spectrum antibacterial effect. The traditional plate counting method is widely used for quantitative detection of viable bacteria and spores but has some disadvantages such as being laborious and time-consuming (at least 24–48 h). This study aimed to develop a new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of B-9987. The specific primers were designed for qPCR amplification based on the conserved region of the bmmA gene (encoding a malonyl CoA-ACP transacylase) of B-9987. According to the characteristic that propidium monoazide (PMA) dye can distinguish viable and dead bacteria, the optimal PMA concentration of 10 μg/ml and optimal exposure time of 10 min were achieved under PMA treatment conditions. The B-9987 spores' genomic DNA was successfully extracted after the spore coat was removed and spore germination was induced. The quantification limits of the PMA-qPCR method were determined for viable B-9987 bacteria, spores in pure culture, and spores in marine Bacillus wettable powder (marine Bacillus WP) and were 1.5 × 103 CFU/ml, 6.5 × 102 CFU/ml, and 103 CFU/ml, respectively. Compared with the qPCR method, the PMA-qPCR method could sensitively detect viable bacteria in the viable/dead bacterial mixture. In this study, the developed PMA-qPCR method was found to have excellent sensitivity and specificity in the context of a pure culture of B-9987 strain, which could accurately and rapidly detect viable B-9987 bacteria within 3–4 h and viable B-9987 spores in marine Bacillus WP within 4–6 h.

Evaluation of phenotypic methods for detection of polymyxin B-resistant bacteria

Determination of sensitivity to polymyxins has always been a challenge, especially in clinical laboratory routines. This study evaluated two rapid, simple, and inexpensive phenotypic methods to test polymyxin B (PMB) susceptibility in Enterobacterales and non-fermenting Gram-negative bacilli. One hundred isolates were used in the tests. The isolates were collected in three hospitals in southern and southeastern Brazil from 1995 to 2019. We compared broth microdilution (reference method) with the broth disk elution test and modified drop test, using polymyxin B -disk or PMB -powder in 2 concentrations (12 and 16 μg/ml). For the broth disk elution and modified drop test with the concentration of 12 μg/ml, categorical agreement values exceeded 90%. The modified drop test with a concentration of 12 μg/ml and broth disk elution may be excellent for initial screening of polymyxin-resistance in laboratory routines. Moreover, these methods are simple and use inexpensive supplies, and may optimize therapeutic decisions.

Lateral flow assays (LFA) for detection of pathogenic bacteria: A small point-of-care platform for diagnosis of human infectious diseases.

Up-to-date diagnostics is globally improved by point-of-care testing (POCT) analysis and bedside research works. Development in POCT analysis has been provided mostly by forward-looking engineering technology for biosensing and sensing assessments. Lately, lateral flow assays (LFAs) have attracted a lot of interest as a result of their noteworthy benefits including cost-effectiveness, better portability, being operator friendly and rapid detection. This technique has been employed broadly for monitoring diverse biomarkers linked to ultrasensitive detection of pathogenic bacteria, ecological monitoring, consumer protection, and infectious diseases. LFA analyses established on qualitative and optical outcomes have boosted the objectivity and data efficiency of the assessments. Therefore, developing novel methods with the capability of providing reliable and quantitative information regarding a target analyte in a model and preserving the qualities of LFAs is of great necessity. In this review, the main principles of LFAs, challenges, and prospects for more development in this field in sensing pathogenic bacteria have been summarized. Subsequently, visually-read LFAs improvement to further progressive platforms have been explored by considering the prospects of this very flexible method for ultrasensitive detection of pathogenic bacteria. In addition, novel labeling methodologies, electrochemical and optical transducers are described. Also, recent developments in these detection methods elements in combination with other considered approaches have been highlighted.

Validation of the Soleris®Enterobacteriaceae Method for Detection of Enterobacteriaceae in Dried Cannabis Flower: AOAC Performance Tested MethodSM 121901.

The Soleris®Enterobacteriaceae vial is a growth-based, automated method for detection of bacteria of the family Enterobacteriaceae in foods and other sample types including nutraceuticals and cosmetics. The Soleris method is used in a "dilute-to-specification" or threshold manner, in which a result is scored as positive or negative around a predetermined cutoff (in CFU/g) established by the dilution and volume of sample homogenate tested. The Soleris method was granted AOAC Performance Tested MethodSM (PTM) status for select foods after successful completion of a validation study (PTM 121901). The objective of this study was to validate the method for the detection of Enterobacteriaceae in dried cannabis flower [>0.3% delta-9-tetrahydrocannabinol (THC)]. The matrix study included comparison of Soleris method presumptive results to confirmation from the Soleris vials, and comparison of the Soleris confirmed results to those of the ISO 21528-2:2017 colony count method. Test materials at four different levels of contamination ranging from 7.8 to 3500 CFU/g were tested at three dilutions, corresponding to test thresholds. Probability of detection analysis at P < 0.05 showed there were no significant differences between Soleris presumptive and confirmed results, and no significant differences between Soleris confirmed and ISO 21528-2:2017 results. The results provided evidence that the Soleris Enterobacteriaceae test is an accurate method for detection of Enterobacteriaceae in dried cannabis flower. The Soleris Enterobacteriaceae method provides cannabis industry QC personnel with an effective method for analysis of dried cannabis flower and produces results in 20-24 h.

Validation of the Soleris® Coliform Method for Detection of Coliform Bacteria in Dried Cannabis Flower: AOAC Performance Tested MethodSM 010302.

The Soleris® Coliform Vial is a growth-based, automated method for detection of coliform bacteria in foods and other consumer products such as nutraceuticals and cosmetics. The method was granted AOAC Performance Tested MethodSM certification for select foods after successful completion of a validation study. The objective of the current study was to validate the Soleris coliform method for use with dried cannabis flower (>0.3% delta 9-tetrahydrocannabinol). A comparative matrix study was performed in which naturally contaminated dried cannabis flower was tested with the Soleris coliform method and with the U.S. Food and Drug Administration Bacteriological Analytical Manual solid medium method. Multiple lots of dried cannabis flower were obtained, pre-screened for coliforms, and blended to produce test materials at four different contamination levels ranging from 4.5 to 1600 CFU/g. Each material was tested at three different Soleris detection threshold levels determined by the dilution used to inoculate the Soleris vials. Probability of detection analysis was performed to determine if differences in the number of positive results obtained with the two methods were significant. For all four dried cannabis flower materials, at all three Soleris test thresholds, there were no significant differences in the number of positive results obtained with the Soleris and cultural plating methods as determined by probability of detection analysis at P < 0.05. The Soleris coliform test is an accurate method for detection of coliform bacteria in dried cannabis flower. The Soleris coliform method provides cannabis industry QC personnel with an effective method for analysis of dried cannabis flower and produces results in 18-24 h.

Target recognition triggered CRISPR-Cas12a assisted allosteric scaffold for sensitively analyzing bacterial infection after dental implantation

Accurate identification and sensitive quantification of infected bacteria after dental implant both play crucial roles in early-diagnosis of bacterial infection and guiding medicine applications. Herein, we propose a sensitive and accurate bacteria detection method based on CRISPR-Cas12a system assisted allosteric scaffold. In the method, the allosteric scaffold takes the responsibility of specifically identifying target bacteria and inducing CRISPR-Cas12a based signal amplification. Eventually, the method exhibits a wide detection range from 6 × 106 cfu/mL to 6 × 102 cfu/mL with the limit of detection (LOD) of 47 cfu/mL. Furthermore, the established approach also possesses a high specificity due to high selectivity of aptamer and robust accuracy in recognizing double strand DNA by CRISPR-Cas12a system. We believe that this work can provide new strategies in the field of diagnosing bacterial infections after dental implantation.

Dual-aptamer-based enzyme linked plasmonic assay for pathogenic bacteria detection

Development of rapid, sensitive, and selective method for pathogenic bacteria detection is of great importance for food safety, medical diagnostic, and environmental monitoring. Currently, most techniques for low numbers of bacteria detection require advanced instrumentation or skilled operators. Herein, we present a facile colorimetric detection platform for bacterial detection using Ag nanoplates as chromogenic substrate, which takes advantages of the high specificity and affinity of aptamer and the ability of catalase to hydrolyze H2O2 that can etch Ag nanoplates. By introducing catalase to the sandwich structure composed by dual-aptamer recognition strategy, bacteria detection signal is converted to the peak shift of LSPR and colorimetric change. This proposed method allows a fast naked-eye detection of S. aureus at the concentration of 60 CFU/mL based on the combination of streptavidin-biotin system and inherent sensitivity of plasmonic Ag nanoplates. Owing to the high selectivity and sensitivity, as well as the low-cost and good adaptability, this plasmonic assay is expected to be suitable for pathogenic bacteria detection in resource-limited settings.

The Recent Original Perspectives on Nonculture-Based Bacteria Detection Methods: A Comprehensive Review.

Foodborne diseases that are primarily caused by pathogenic bacteria are of major public health concern globally. One of the key strategies in minimizing and controlling the risk of contamination of food with such pathogens requires establishing effective detection and tracking methods of zoonotic bacteria. Although culture-based pathogen detection methods are developed and widely used by the industry traditionally, nonculture-based zoonotic bacteria detection methods are now more widely investigated and used owing to the recent developments in nucleic acid and immunological-based detection methods. These rapid detection methods provide the opportunity of acquiring real-time test results and high-throughput screening of a large number of samples at a time. One of the key aspects of rapid detection methods is the development of effective sample processing methods as food samples are heterogeneous and highly complex in composition. In this review, variety of sample processing methods, in terms of nonspecific and target-specific sample processing as well as thorough overview of recent developments in nonculture-based bacteria detection methods are presented.

Rapid Detection and Antimicrobial Susceptibility Testing of Pathogens Using AgNPs-Invertase Complexes and the Personal Glucose Meter.

Rapid detection of pathogens and assessment of antimicrobial susceptibility is of great importance for public health, especially in resource-limiting regions. Herein, we developed a rapid, portable, and universal detection method for bacteria using AgNPs-invertase complexes and the personal glucose meter (PGM). In the presence of bacteria, the invertase could be released from AgNPs-invertase complexes where its enzyme activity of invertase was inhibited. Then, the enzyme activity of invertase was restored and could convert sucrose into glucose measured by a commercially PGM. There was a good linear relationship between PGM signal and concentration of E. coli or S. aureus as the bacteria model with high sensitivity. And our proposed biosensor was proved to be a rapid and reliable method for antimicrobial susceptibility testing within 4 h with consistent results of Minimum Inhibitory Concentrations (MICs) testing, providing a portable and convenient method to treat infected patients with correct antibiotics and reduce the production of antibiotic-resistant bacteria, especially for resource-limiting settings.

Wulff-type boronic acid-functionalized quantum dots for rapid and sensitive detection of Gram-negative bacteria

Bacterial infection is a major threat to human health. However, traditional bacteria detection methods need long time and specific conditions. This work reports a fluorescent Gram-negative bacteria probe based on CdSe/CdS/ZnS quantum dots (QDs). The interaction between L-cysteine (Cys) - 4-aminophenylboronic acid (APBA) functionalized QDs (QDs/Cys-APBA) and bacteria outer surfaces lipopolysaccharide induces rapid quenching of the fluorescence of QDs. Escherichia coli and P. Aeruginosa, two typical Gram-negative bacteria, can be detected in 30 min and with a lower limit of detection (LOD) of 58 and 97 CFU/mL, respectively. The probe with the Wulff-type boronic acid structure formed by Cys and APBA is very sensitive to Escherichia coli over a relative wide range of pH from 5.0 to 9.0. It retains high selectivity and sensitivity in the presence of other Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis. Its practical potential is shown for estimating the degree of Escherichia coli contamination in cow milk. Meanwhile, bacteria growth is hardly affected by the presence of the QDs/Cys-APBA in the detection process, showing the robustness of the QDs/Cys-APBA as a probe for bacteria detection. This study provides an efficient route for rapid and highly sensitive pathogen diagnosis.

Sandwich method-based sensitivity enhancement of Ω-shaped fiber optic LSPR for time-flexible bacterial detection

The development of rapid and sensitive detection methods for pathogenic bacteria is crucial for the therapy and prevention of related diseases. However, the rapid and ultrasensitive assays are difficult to be realized simultaneously. To solve the problem, a sandwich method based on Ω-shaped fiber optic localized surface resonance (Ω-FOLSPR) was constructed, where poly adenine-tailed aptamer (PolyA-apt) and SH modified gold nanoparticles tags (AuNPs tags) were chosen as the capturing aptamer and amplifying tags, respectively. The small AuNPs were modified on the surface of fiber-optic (FO) rapidly, which saved the preparation time. Then, the PolyA-apt was modified on the AuNPs surface to capture the bacteria effectively due to its ability to adjust the density and conformation of aptamer on the AuNPs surface. Finally, the large AuNPs tags were used to generate intense signal enhancement. It is found that the sandwich method enables the unique characteristic of a time-dependent sensitivity enhancement. Specifically, the LOD of 108.0 CFU/mL and 7.4 CFU/mL was achieved with the analysis time of 10 min and 100 min, respectively. Besides, the Ω-FOLSPR sensor exhibits excellent selectivity against the other bacteria and good performance for detecting the spiked and natural samples. This sandwich method provides a time-flexible strategy due to the combination of effective signal amplification and real-time analysis for bacterial detection, displaying great potential for practical bacterial detection.

A sensitive visual method for onsite detection of quarantine pathogenic bacteria from horticultural crops using an LbCas12a variant system

Pre-planting testing of seeds and plantlets for the existence of quarantine pathogens is an important phytosanitary measure. The CRISPR-mediated molecular diagnostic methodologies are being developed for pathogens detection, but many challenges remain. Here, we profiled an engineered Crispr/LbCas12a variant (LbCas12a-5M) that has more robust trans-cleavage activity and a wider PAM sequences (TNTN) preference than wild type. We developed a procedure for screening specific sequences of bacterial plant pathogens, and the designed species-specific crRNA displayed no cross-reactions with other bacterial species. Combined with a simple extraction of bacterial DNA, an LbCas12a-5M-based visual detection technique was established and optimized for detecting quarantine pathogens Erwinia amylovora and Acidovorax citrulli with detection limits up to 40 CFU/reaction and a sensitivity consistent with qPCR assay. This protocol was faster and simpler than qPCR, requiring 40 min or less from sample preparation. We further validated the potential application of the method by showing that it can be used for rapid and accurate diagnosis of A. citrulli on seeds of watermelon, with 100% agreement with the results of qPCR assay. The developed method simplifies the detection of pathogens and provides cost-effective countermeasures to quarantine interventions.

A rapid and efficient DNA isolation method for qPCR-based detection of pathogenic and spoilage bacteria in milk

The objective of this study was to find an efficient, rapid, simple, and cost-effective method of pretreating raw milk samples to produce PCR-ready DNA for subsequent microbial detection using the strains of eight bacterial species. A total of 17 in-house protocols and three commercial kits were evaluated in three steps from scientific, practical, and economic perspectives. The results showed that an in-house procedure involving Triton X-100-based pretreatment and an inhibitor removal resin was superior to all other methods tested in terms of DNA yield, sensitivity, ease of sample handling, time efficiency, and cost per sample. Overall, this simplified preanalytical protocol was shown to have a great potential for use in rapid detection of dairy-related bacterial species, thereby enabling early intervention in the food chain and thus reducing the risk of negative economic and health outcomes. • Methods of pretreating raw milk samples to yield PCR-ready bacterial DNA were tested. • Of 20 procedures, a Triton X-100-based in-house protocol showed the best results. • This approach has a great potential for use in detection of dairy-related bacteria. • With further optimization, the protocol may be included in milk quality guidelines.

High-Throughput Flow-Through Direct Immunoassays for Targeted Bacteria Detection.

Regulatory authorities require analytical methods for bacteria detection to analyze large sample volumes (typically 100 mL). Currently only the Membrane Filtration and the Most Probable Number assays analyze such large volumes, while other assays for bacteria detection (ELISA, lateral flow assays, etc.) typically analyze volumes 1000 times smaller. This study describes flow-through direct immunoassays (FTDI), a new methodology for the targeted detection of bacteria in liquid samples of theoretically any volume. Flow-through direct immunoassays are performed in fluid-permeable microwells (e.g., wells of a filter well plate) that have a membrane on their bottom where the bacteria are trapped before their detection using a direct immunoassay. Two versions of FTDI assays for the detection of E. coli in 10 mL of sample were developed. A rapid FTDI assay that can be completed in less than 2.5 h can detect E. coli bacteria in levels down to 17 CFU/mL, and an ultrasensitive FTDI assay that employs an additional bacteria culturing step to boost the sensitivity can detect E. coli bacteria in levels lower than 1 CFU/mL in less than 5.5 h. All the steps of the assays, including the immunoassay steps, the culturing step, and the analytical signal measurement step are performed inside the well plate to decrease the chance of contamination and ensure a safe, easy process for the user. The assays were assessed and validated in tap water, river water, and apple juice samples, and the results suggests that the assays are robust, precise, and accurate. When the assays are performed in 96-well filter plates, a filter well plate vacuum manifold and a multichannel peristaltic pump are also used, so multiple samples can be analyzed in parallel to allow high-throughput analysis of samples.

The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification and PCR Methods in Detection of Foodborne Microorganisms: A Systematic Review and Meta-Analysis.

Background:The loop-mediated isothermal amplification (LAMP) method is frequently used for identifying many microorganisms. The present review aimed to evaluate the sensitivity and specificity of LAMP method for detection of food-borne bacteria and to compare these features with those of polymerase chain reaction (PCR), as an alternative molecular diagnostic procedure, and with cultivation method, as the gold standard method.Methods:The literature was searched in electronic databases (PubMed, Scopus, Web of Science, and EMBASE) for recruiting publications within Jan 2000 to Jul 2021. We used the combinations of keywords including foodborne disease, LAMP, PCR, Loop-mediated isothermal amplification, and polymerase chain reaction. Meta-analysis was used to adjust the correlation and heterogeneity between the studies. The efficiency of the methods was presented by negative likelihood ratio, positive likelihood ratio, sensitivity, specificity, and odds ratio using forest plots. A P-value less than 0.05 was considered as statistical significance cut off. The confidence intervals were presented at the 95% interval.Results:Overall, 23 relevant studies were analyzed. The sensitivities of LAMP and PCR methods were estimated to be 96.6% (95% CI: 95.0–97.7) and 95.6% (95%CI: 91.5–97.8), respectively. The specificities of LAMP and PCR were also estimated to be 97.6% (95%CI: 92.6–99.3) and 98.7% (95%CI: 96.5–99.5), respectively.Conclusion:The specificities of LAMP and PCR assays were determined by comparing their results with cultivation method as the gold standard. Overall, the specificity of both PCR and LAMP methods was low for detection of fastidious bacteria. Nevertheless, LAMP and PCR methods have acceptable specificities and sensitivities, and their application in clinical practice necessitates more studies.