Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay.
ABSTRACT: BACKGROUND: Hepatitis B virus (HBV) is an important blood-borne pathogen that causes hepatic inflammation and can lead to liver cirrhosis and hepatocellular carcinoma. Conventional methods of HBV detection are time consuming and require highly trained personnel and elaborate equipment. This report describes the development of a rapid, simple, specific, and sensitive loop-mediated isothermal amplification assay (LAMP) for detection of HBV genotypes A, B, C, D, E, and F in blood samples. METHODS: HBV standard plasma panels and clinical donor plasma specimens were used for the development and validation of the LAMP assay. Amplification was performed at 60°C for 60 minutes using extracted DNA or heat-treated plasma specimens without DNA extraction. The assay was evaluated for its ability to detect various HBV genotypes and for its sensitivity, specificity, and time-point of detection. RESULTS: The LAMP assay detected HBV genotypes A-F and demonstrated a sensitivity of 10-100 IU per reaction of HBV DNA. The assay also detected 69 of 75 (92%) HBV-positive donor plasma specimens tested and demonstrated a specificity of 100%. CONCLUSIONS: These results demonstrate that our HBV-LAMP assay is rapid, sensitive and specific, and capable of detecting the major HBV genotypes. This assay could be used in clinical point-of-care settings, mainly in endemic and resource-limited environments for HBV diagnostics, donor screening, epidemiological studies, and therapeutic monitoring of patients undergoing antiviral treatment.
Project description:Recently, many studies have demonstrated the significant advantages of loop-mediated isothermal amplification (LAMP) based methods over serological tests and PCR for rapid detection of microbial pathogens. Here, a rapid LAMP assay was developed to detect the hepatitis B virus (HBV) from DNA, and particularly, blood samples from infected patients using a commercially available master mix and portable real-time fluorometer. The final optimized fluorescence-based LAMP assay provided significant amplification time of less than 15 minutes compared with over 1 hour for PCR and an opened tube LAMP system described previously. Results indicated that fluorescence-based LAMP assay was more sensitive than PCR as a rapid, sensitive, efficient, and highly reliable approach for rapid detection of HBV.
Project description:Specific genotypes of hepatitis B virus (HBV) are increasingly recognized for their clinical significance and association with particular viral mutations. Although many HBV genotyping methods exist, there has been no standardized or commercially available method for direct molecular typing of the HBV genome. A newly available line probe assay (INNO-LiPA HBV Genotyping assay; Innogenetics N.V., Ghent, Belgium) that allows the identification of HBV genotypes A to G was assessed by comparison with pre-S1/pre-S2 sequence analysis of the isolates in 188 serum specimens. All seven genotypes were detected by the line probe assay (LiPA), and complete concordance between LiPA and sequence analysis was observed for 152 specimens (81%). LiPA was able to detect 19 mixed genotype infections not detected by amplicon sequencing, which for the most part were confirmed by cloning and sequencing of the pre-S1/pre-S2 amplicon. Four specimens had discrepant results between the two methods, and five specimens had indeterminate results by LiPA. The HBV DNA in four specimens was unable to be amplified by the nested INNO-LiPA HBV DR amplification primers; however, the HBV DNA in six specimens unable to be genotyped by sequencing was clearly genotyped by LiPA. The INNO-LiPA HBV Genotyping assay appears to be useful for the rapid genotyping of HBV, particularly for the sensitive detection of mixed genotype infections.
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:BACKGROUND:JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunosuppressed patients. PML usually has a poor prognosis. Detection and quantification of the JCV genome in cerebrospinal fluid (CSF) is an efficacious tool for the diagnosis and management of PML, for which proper therapeutic interventions are required. METHODS:A loop-mediated isothermal amplification (LAMP) assay was applied for the quantitative detection of JCV. The LAMP assay was evaluated for the efficacy in diagnosis of PML in comparison with the TaqMan-based quantitative real-time PCR (qPCR) assay using 153 CSF specimens collected from patients with suspected PML. RESULTS:The LAMP assay showed no cross-reactivity against other polyomavirus plasmids, viral DNA, and viral RNA, which causes encephalitis, and detected 1 copy of the standard DNA per reaction. Among 50 qPCR-positives, 42 specimens (containing JCV genome ranged from 3.2?×?100 to 3.2?×?106 copies/reaction) showed positive reactions and 8 specimens (containing 0.9 to 19.9 copies/reaction) showed negative in the LAMP assay. Furthermore, 3 of 103 qPCR-negative specimens showed positive reactions in the LAMP assay. The sensitivity, specificity, positive predictive value, and negative predictive values of the LAMP assay were 84% (42/50), 97% (100/103), 93% (42/45), and 93% (100/108), respectively. The kappa statistic was 0.83. The JCV loads determined by the LAMP assay showed a strong positive correlation with those determined by the qPCR assay for 33 specimens with copy numbers of ?1 copies/reaction (r?=?0.89). Additionally, the LAMP assay could monitor the JCV genome copy number in CSF for sequential samples equivalently to qPCR assay. CONCLUSIONS:The newly developed LAMP assay is highly specific against JCV and detect the JCV genome in the sample DNA containing 20 or more copies of JCV genome per reaction with 100% sensitivity (n?=?29), which corresponds to ?3?×?103 copies/mL of CSF. The LAMP assay is useful for the diagnosis and offers valuable information for the evaluation and management of PML in the clinical setting.
Project description:Haemophilus influenzae type b (Hib) is one of the leading causes of meningitis in developing countries. To establish and evaluate a novel loop-mediated isothermal amplification (LAMP) assay for Hib, we designed a LAMP primer set targeting the Hib-specific capsulation locus. LAMP detected 10 copies of purified DNA in a 60-min reaction. This indicated that the detection limit of LAMP was >100-fold lower than the detection limits of both a PCR for the detection of bexA and a nested PCR for Hib (Hib PCR). No H. influenzae, other than Hib or control bacteria, was detected. Linear determination ranged from 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the Hib LAMP assay using a set of 52 randomly selected cerebrospinal fluid (CSF) specimens obtained from children with suspected meningitis. For comparison, the CSF specimens were tested using a conventional Hib PCR assay. Hib was detected in 30 samples using LAMP and in 22 samples using the Hib PCR assay. The Hib PCR showed a clinical sensitivity of 73.3% and a clinical specificity of 100% relative to the Hib LAMP assay. These results suggest that further development and evaluation of the Hib LAMP will enhance the global diagnostic capability for Hib detection.
Project description:Background:To improve urinary tract infection detection, we evaluated the specificity and sensitivity of Loop-mediated isothermal Amplification Method (LAMP) for detection of the Eschericia coli (E. coli) in urine samples, for the first time. Methods:Primers were designed to target the malB gene of Escherichia coli. LAMP assay was performed on urine specimens collected from patients with urinary tract infection symptoms. Results:As expected, LAMP was more specific and sensitive than direct microscopic tests. LAMP assay showed the best detection limit of DNA copies with 1.02 copies. Conclusion:LAMP method offers several advantages in terms of sensitivity, rapidness and simplicity for detection of E. coli infection in urine samples. The LAMP method would be highly suitable for the early detection of the UTIs and also comfort quick diagnosis of UTI in clinical laboratories with limited equipment.
Project description:BACKGROUND: Hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) is very common in China. It is difficult to distinguish between EV71 and coxsackievirus A16 (CVA16) infections in clinical HFMD patients. Routine laboratory diagnosis of EV71 infection is time-consuming and requires expensive instruments. In this study, we have developed a one-step, single tube, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of EV71. METHODS: Six primers that can recognize 6 distinct regions on the VP2 gene of EV71 were designed for RT-LAMP assay. The amplification was completed by incubating all reagents in a single tube with reverse transcriptase and Bst DNA polymerase under the isothermal condition (60°C) for 60 min, and could be evaluated by using GoldView staining under a handheld ultraviolet torch lamp or electrophoresis analysis. RESULTS: A total of 123 specimens collected from suspicious patients with HFMD were simultaneously detected by RT-LAMP and PCR fluorescence probing assay. The RT-LAMP amplified products containing EV71 were digested by HinfI and TaqI restriction endonucleases; in contrast, non-specific products with CVA16, coxsackievirus A4 and coxsackievirus B3 could not be detected in RT-LAMP assay. Meanwhile, RT-LAMP assay could amplify EV71 virus with a detection limit of 1 PFU/ml within 60 min. Compared with PCR fluorescence probing assay, RT-LAMP assay exhibited 98.4% identity during the detection of EV71 viral RNA without the missing of positive samples. CONCLUSION: Our results indicated that RT-LAMP is a rapid, sensitive, specific and accurate method for the detection of EV71 in clinical specimens. Therefore, this developed method has potential application for rapid and comprehensive surveillance for EV71 infection, especially in developing country.
Project description:Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) ?-lactamase-producing strains are of growing concern. Several genotypes of the GES ?-lactamase gene (bla GES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of bla GES and another LAMP method to discriminate carbapenemase genotypes of bla GES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting bla GES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting bla GES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected bla GES with high sensitivity in all DNA-spiked samples; PCR did not detect bla GES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing bla GES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two bla GES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES ?-lactamase gene detection assay using the LAMP method. Our new assays effectively detect bla GES and critical unique mutations.
Project description:In this study, we report a simple, low-cost surface plasmon resonance (SPR)-sensing cartridge based on a loop-mediated isothermal amplification (LAMP) method for the on-site detection of the hepatitis B virus (HBV). For LAMP detection, a SPR based LAMP sensing system (SPRLAMP) was constructed, including a novel SPRLAMP sensing cartridge integrating a polymethyl methacrylate (PMMA) micro-reactor with a polycarbonate (PC)-based prism coated with a 50 nm Au film. First, we found that the change of refractive index of the bulk solution was approximately 0.0011 refractive index (RI) units after LAMP reaction. The PC-based prism's linearity and thermal responses were compared to those of a traditional glass prism to show that a PC-based prism can be used for SPR measurement. Finally, the HBV template mixed in the 10 ?l LAMP solution could be detected by SPRLAMP system in 17 min even at the detection-limited concentration of 2 fg/ml. We also analyzed the correlation coefficients between the initial concentrations of HBV DNA templates and the system response (?RU) at varying amplification times to establish an optimal amplification time endpoint of 25 min (R(2)=0.98). In conclusion, the LAMP reaction could be detected with the SPRLAMP sensing cartridge based on direct sensing of the bulk refractive index.
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.