Rapid detection of herpes simplex virus DNA in cerebrospinal fluid: comparison between loop-mediated isothermal amplification and real-time PCR

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and speed under isothermal conditions. To evaluate the usefulness of LAMP for diagnosing central nervous system infection with herpes simplex virus (HSV), we compared the LAMP method with real-time PCR, using samples that were previously tested by nested PCR. We examined 69 cerebrospinal fluid (CSF) samples from patients suspected of having HSV infection of the central nervous system. The results of the real-time PCR analysis and nested PCR assay were in complete accord. When nested PCR was regarded as standard, the sensitivity of LAMP was 81%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 90%. Although further improvement is necessary for the wide spread use, the LAMP method might be applicable to diagnosis of HSV infection of the central nervous system.     

Serum Epstein-Barr virus DNA load in primary Epstein-Barr virus infection

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. During acute, lytic phase of infection, viral DNA can also be detected in serum. In the present study, the diagnostic utility of EBV DNA detection and quantitation in serum in primary EBV infection was investigated. The level of EBV DNA in the serum of 98 immunocompetent patients aged 1-47 years with symptomatic, antibody-confirmed EBV primary infection was assessed using a quantitative real-time PCR assay. The association between viral load and time after onset of disease, age and clinical and laboratory data was investigated. Quantitative PCR detected EBV DNA in 93 of 98 samples (94.9%), and the measured viral loads ranged from 3.8 x 10(1) to 6.6 x 10(4) copies/ml. EBV DNA detection exhibited a sensitivity of 94.9% and a specificity of 97.4% for primary EBV infection. EBV DNA was always detectable until day 12 after onset of symptoms, whereas no further positive PCR results were found after a period of 22 days after onset of disease. Detection of EBV DNA also showed a clearer association with the clinical manifestation of disease than the presence of EBV specific VCA IgG antibodies of low avidity. EBV DNA load was found to correlate inversely with the time after onset of disease (P < 0.001), and higher viral load levels were detected in younger (P = 0.009) and in hospitalized patients (P = 0.038). The results indicate that real-time PCR is a reliable tool for diagnosis of primary EBV infection early in the course of disease. In addition, EBV DNA detection may serve as a useful diagnostic supplement in serologically indeterminate EBV infections.     

A rapid and highly reliable field-based LAMP assay of canine parvovirus

Loop-mediated isothermal amplification (LAMP) is known as a rapid and reliable alternative to conventional single-step or nested PCR for detection of genomic DNA of various pathogens in clinical samples. In this study, LAMP assay was developed for canine parvovirus (CPV) and compared with single-step and nested PCR assays. Out of 50 fecal samples from dogs clinically suspected for CPV infections, 19 were found positive by single-step PCR, 22 by nested PCR and 26 by LAMP. LAMP products were subjected to restriction analysis and sequencing to check their specificity. LAMP assay turned out to be a rapid and fairly reproducible method, did not amplify other common canine pathogens and was more sensitive than nested PCR assay. Therefore, it can be regarded as a highly reliable method for routine field diagnosis of CPV infection. Keywords: canine parvovirus; nested polymerase chain reaction; loop-mediated isothermal amplification; sensitivity; specificity.     

Rapid detection of varicella-zoster virus infection by a loop-mediated isothermal amplification method

The reliability of varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) was evaluated for rapid diagnosis of viral infection. VZV-specific primers only amplified VZV DNA; no LAMP products were observed in reactions performed with other viral DNA templates. The specificity of this method was confirmed by two independent determinations, agarose gel electrophoresis and a turbidity assay. The sensitivity of VZV LAMP, determined by agarose gel electrophoresis, were 500 copies/tube. Detection using the turbidity assay, however, gave a sensitivity of 1,000 copies/tube. After these initial validation studies, reliability of VZV LAMP was evaluated for the detection of viral DNA in clinical specimens. Thirty-two swab samples collected from patients with vesicular skin eruptions were tested for VZV DNA. VZV was confirmed in sample numbers 10-32 by VZV real-time PCR, a previously established technique. VZV LAMP products were detected using turbidity from samples 13 to 32 (sensitivity; 87.0%, specificity; 100%, positive predictive value; 100%, negative predictive value; 75%). Although low levels of VZV DNA could be detected in the three samples exhibiting divergent results (samples numbers 10-12), no VZV LAMP product was detected in these samples, indicating a higher detection limit for this assay. Requirement of a DNA extraction step in the VZV LAMP method was examined in next experiment. The turbidity assay detected a VZV LAMP product in all of the 20 positive swab samples (samples numbers 13-32), regardless of DNA extraction.     

Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi

A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/μl of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.     

Loop-mediated isothermal amplification assay for the diagnosis of retinitis caused by herpes simplex virus-1

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of herpes simplex virus 1 (HSV-1). The specificity of the assay was tested using DNA extracted from HSV-1-infected rabbit corneal epithelium cultures, HSV-2 grown on Vero cell line, cytomegalovirus (CMV) (AD-169), varicella zoster virus (VZV) (Oka-vaccine), adenovirus, Aspergillus flavus and Staphylococcus aureus. The specificity of LAMP was confirmed by bidirectional sequencing of the amplicons. The sensitivity of the LAMP assay was tested using different concentrations of HSV-1 DNA. To evaluate the application of the LAMP assay in clinical diagnosis, we tested vitreous samples from 20 patients with suspected viral retinitis using LAMP and real-time PCR for HSV-1. The LAMP primers amplified only HSV-1 DNA; no LAMP products were detected with the DNAs of HSV-2, CMV, VZV, adenovirus A. flavus and S. aureus. The sequences of the positive HSV-1 LAMP products perfectly (99–100%) matched the HSV-1 sequences deposited in the GenBank database. LAMP is as sensitive as real-time PCR, with the lowest detection limit being 10 copies/μL of HSV-1 DNA. Of the 20 patients with suspected viral retinitis, four tested positive for HSV-1 using real- time PCR and LAMP. A 100% concordance was observed across the two methods. The LAMP assay is a rapid, highly specific and sensitive method for the diagnosis of retinitis caused by HSV-1.     

Rapid detection of bovine herpesvirus 1 in bovine semen by loop-mediated isothermal amplification (LAMP) assay

Bovine herpesvirus 1 (BoHV-1) is the most common viral pathogen found in bovine semen, causing numerous reproductive disorders leading to economic losses to the cattle industry. For rapid detection of BoHV-1 in bovine semen, in this study, we applied a loop-mediated isothermal amplification (LAMP) assay. The assay could be completed within 90 min, including total DNA isolation, target amplification, and visual interpretation of positive or negative results with the naked eye. The assay detected as little as 10 fg of BoHV-1 DNA per reaction. The analytical sensitivity of the assay was 0.2 TCID₅₀ BoHV-1 per reaction, which was 100 times more sensitive than conventional PCR and comparable to TaqMan real-time PCR. The applicability of the assay was assessed by analysing 118 semen samples collected from breeding bulls. On comparison with TaqMan real-time PCR, the LAMP assay had a diagnostic sensitivity of 97 %, specificity of 100 %, and accuracy of 99.2 % for detection of BoHV-1 in bovine semen. The LAMP assay developed in this study is a rapid, sensitive, and cost-effective alternative for detection of BoHV-1 in bovine semen.     

Rapid and sensitive diagnosis of Acanthamoeba keratitis by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of Acanthamoeba. The sensitivity of the LAMP assay was tested using different copies of positive DNA. The specificity of the assay was tested using DNA extracted from Acanthamoeba, Pseudomonas aeruginosa, Candida albicans, herpes simplex virus-1 and human corneal epithelial cells. Its effectiveness was evaluated and compared with culture, corneal smear examination and real-time PCR in corneal samples from mice with Acanthamoeba keratitis. We also tested three corneal samples from patients with suspected Acanthamoeba or fungal infection using LAMP. Loop-mediated isothermal amplification was confirmed to be very sensitive, with the lowest detection limit being ten copies/tube of Acanthamoeba DNA. The LAMP primers only amplified Acanthamoeba DNA. During the development of Acanthamoeba keratitis in mice, almost all of the positive rates of LAMP at each time post-infection were higher than those of culture or corneal smear examination. The total positive rate of LAMP was significantly higher than those of culture and corneal smear examination (p <0.05), whereas the sensitivities of LAMP and real-time PCR were comparable. However, the trends of positive change in these different test methods were generally similar. Of the three clinical corneal specimens, two with suspected Acanthamoeba keratitis tested positive for Acanthamoeba using LAMP along with culture or corneal smear examination, whereas the other suspected fungal keratitis tested negative. The LAMP assay is a simple, rapid, highly specific and sensitive method for the diagnosis of keratitis caused by Acanthamoeba.     

Rapid detection of orf virus by loop-mediated isothermal amplification based on the DNA polymerase gene

At present, there are no effective antiviral treatments available for contagious ecthyma, and rapid diagnosis is therefore critical for effective control of the disease. Recently, the invention of a novel LAMP technique that can rapidly amplify nucleic acids with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic acid-based diagnostic tests and has made on-site diagnosis possible. To establish a flexible loop-mediated isothermal amplification (LAMP) assay for the rapid detection of orf virus, two pairs of primers, including outer primers F3/B3 and inner primers FIP/BIP, were designed to amplify the DNA polymerase gene. Optimal time and temperature conditions for LAMP were found to be 45 min and 62 °C, respectively. The LAMP assay was shown to be specific, with no cross-reactivity with sheeppox virus, goatpox virus, avian molluscum roup virus or vesicular stomatitis virus. Additionally, the sensitivity of the LAMP method was similar to that of real-time PCR and demonstrated greater sensitivity than a conventional polymerase chain reaction (PCR) assay. To assess the utility of LAMP in the detection of orf virus in clinical samples, a total of 35 samples collected from orf virus-infected sheep and goats were tested using the optimized LAMP assay, real-time PCR, and conventional PCR. Of the samples, 26 were found to be positive by LAMP, and 25 (74.3 %) were positive by real-time PCR, whereas only 18 (51.4 %) were positive by conventional PCR. Our results have shown that the LAMP assay developed in this study can be used for the rapid detection of orf virus.     

环介导的等温扩增与实时荧光PCR检测问号钩端螺旋体的比较

目的 通过比较环介导的等温扩增技术(loop-mediated isothermal amplification,LAMP)与实时荧光PCR( real-time PCR)技术在检测问号钩端螺旋体的特异性及灵敏度方面的差异,寻找一种快速、灵敏且特异性强的问号钩端螺旋体检测方法。方法 根据问号钩端螺旋体lipL41基因序...     

Direct detection of human herpesvirus 6 DNA in serum by the loop-mediated isothermal amplification method.

BACKGROUND: A more rapid and easier method is needed for monitoring human herpesvirus 6 (HHV-6) infections. The loop-mediated isothermal amplification method (LAMP) can detect viral DNA with high specificity, efficiency, and speed under isothermal conditions. LAMP requires only simple equipment that is available in hospital laboratories. OBJECTIVES: We evaluated LAMP as a means of detecting HHV-6 DNA directly from patients' sera. RESULTS: The sensitivity of the HHV-6 LAMP protocol without heat denaturation was 1000 copies/tube; with heat denaturation 10 copies/tube were detected. Three hundred serum samples from children with fever were analyzed. Using HHV-6 isolation as a definition of HHV-6 infection, the sensitivity, specificity, positive predictive value, and negative predictive value of the HHV-6 LAMP method without DNA extraction were 95.5%, 95.2%, 94.0%, and 96.4%, respectively. CONCLUSION: Direct detection of HHV-6 DNA in serum with a modified HHV-6 LAMP could be used for rapid diagnosis of exanthem subitum (ES).     

Loop-mediated isothermal amplification assay for detection of Haemophilus influenzae type b in cerebrospinal fluid

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.     

Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the rapid detection of the major periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The LAMP method amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. In this study, we initially designed the primers for LAMP assays to detect these bacteria and evaluated the specificity and sensitivity of these assays. The specificities of the primers for these bacteria were examined using various oral bacteria and various reaction times. The lower detection limits of the 60-min LAMP reaction without loop primers were 1 microg/tube for P. gingivalis, 10 fg/tube for T. forsythia, and 1 ng/tube for T. denticola. Addition of the loop primers for each bacterium improved the detection specificities and sensitivities by several magnitudes. Furthermore, LAMP assays were applied to the rapid detection of these periodontal pathogens in clinical specimens, and the results were compared with those of conventional PCR detection. The results of the LAMP assays corresponded to those of conventional PCR assays. These results indicate that the LAMP assay is an extremely rapid, highly sensitive, specific method. This method is very useful for the rapid detection of periodontopathic bacteria and the diagnosis of periodontal disease.     

A loop-mediated isothermal amplification method for rapid detection of NDM-1 gene

A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40?min under isothermal conditions at 65°C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1?pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.     

Loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18

A new method was developed for detection of human papillomavirus (HPV) by loop-mediated isothermal amplification (LAMP), which was compared with the polymerase chain reaction (PCR), and real-time PCR for specificity and sensitivity. All initial validation studies with the control DNA proved to be type-specific. In order to evaluate the reliability of HPV type-specific LAMP detecting HPV DNA from clinical samples, tissue specimens were obtained from 27 patients with external genital polypoid lesions. The histologic diagnoses included condyloma acuminatum (n = 21), bowenoid papulosis (n = 2), seborrheic keratosis (n = 2), epidermolytic acanthoma (n = 1), and hairy nymphae (n = 1). HPV-6 DNA and HPV-11 DNA were detected in 18 and 3 of 21 condylomata acuminata, respectively, and there was no simultaneous infection. HPV-16 DNA was detected in one of two bowenoid papuloses. HPV DNA was not detected in the seborrheic keratoses, epidermolytic acanthoma, and hairy nymphae. These results correlated perfectly with those from real-time PCR analysis. Most positive samples contained high copy numbers of HPV DNA. HPV-11 DNA was detected in one case that could not be detected by PCR. The average reaction time was about 59 min. There was a linear correlation between the genome quantity and reaction time to reach the threshold. The LAMP method has an additional advantage as a quantitative method, and is superior in terms of sensitivity, specificity, rapidity, and simplicity, and can potentially be a valuable tool for the detection of HPV DNA.     

Comparison of the illumigene Mycoplasma DNA Amplification Assay and Culture for Detection of Mycoplasma pneumoniae

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.     

Rapid, simple, and sensitive detection of Anaplasma phagocytophilum by loop-mediated isothermal amplification of the msp2 gene

Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis, which is prevalent throughout China. In this study, we describe a rapid, simple, and sensitive loop-mediated isothermal amplification (LAMP) assay targeting the msp2 gene of A. phagocytophilum that is ideal for application in rural areas in China. This assay has the potential to detect A. phagocytophilum early in infection as an alternative to existing methods. A total of 42 suspected cases of infection with A. phagocytophilum, 15 serologically confirmed and 27 probable cases, were analyzed by the msp2 LAMP assay. To validate the accuracy of LAMP, previously established nested-PCR and real-time PCR assays were utilized. The sensitivity of LAMP was 25 copies per reaction (approximately 1,250 copies per ml blood) for A. phagocytophilum, and the assay did not detect false positives among 27 members of the order Rickettsiales and 17 common clinical pathogens. To evaluate the clinical applicability of the LAMP assay, a total of 42 clinical samples were examined. A positive LAMP result was obtained for 12 of the confirmed cases and for 14 of 27 suspected cases, while only 1 confirmed case and 3 cases (2 confirmed cases and 1 suspected case) were detected by nested PCR and real-time PCR, respectively. The LAMP assay described in this study demonstrated a high level of sensitivity comparable with that of nested PCR and real-time PCR for the detection of A. phagocytophilum. This LAMP assay is a valuable method for rapid, cost-effective, and simple detection of A. phagocytophilum in the rural areas of China.     

Development and evaluation of real-time loop-mediated isothermal amplification for hepatitis B virus DNA quantification: a new tool for HBV management.

BACKGROUND: Accurate quantitation of hepatitis B viral load is a critical aspect in screening and monitoring HBV infection. OBJECTIVES: We used loop-mediated isothermal amplification (LAMP) to develop a real-time fluorogenic (RtF-LAMP) protocol to quantitate HBV DNA. Quantitative analysis was obtained by measuring time-to-positive (TTP), a biomarker similar to cycle threshold (Ct) in real-time PCR. STUDY DESIGN: Sensitivity, specificity, reproducibility, and dynamic range for RtF-LAMP were evaluated using molecular and biological standards. Four hundred and two patient samples were then used to compare the performance of RtF-LAMP to a commercial real-time PCR assay (DaAn Gene Co, China). RESULTS: The lower detection limit (LDL) of RtF-LAMP by Probit analysis at the 95% detection level was 210 copies/ml, and the dynamic range was 8 orders of magnitude. The conversion factor for results obtained with the RtF-LAMP assay was 1 IU/ml equals to 4.4 copies/ml. Coefficients of variation (CV) reflected low intra-assay and inter-assay variability (4.24-12.11%). In a large number of serum samples, there was excellent an correlation between RtF-LAMP and real-time PCR (R(2)=0.96). There was a good agreement between the two tests except at the detection cutoff of the real-time PCR assay. CONCLUSION: Our RtF-LAMP protocol appears to be precise, accurate and rapid. It could be a valuable tool for the detection of HBV in large clinical and epidemiological studies.     

Comparison of loop-mediated isothermal amplification, real-time PCR, and virus isolation for the detection of herpes simplex virus in genital lesions

This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions; two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samples testing positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%.     

Sensitive and rapid detection of the new Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification

New Delhi metallo-β-lactamase 1 (NDM-1), which is associated with resistance to carbapenem, was first reported in 2008. A sensitive and rapid molecular assay to detect the plasmid bla(NDM-1) in clinical isolates is needed to control its spread. We describe a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of bla(NDM-1) from pure culture and sputum, urine, and fecal samples. Eight sets of primers were designed to recognize six or eight distinct sequences on target bla(NDM-1), and one set was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for bla(NDM-1) detection were determined. The sensitivity of the LAMP assay for bla(NDM-1) detection in sputum, urine, and fecal samples was also tested. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 70 min at an isothermal temperature of 65°C. The sensitivity of LAMP, with a detection limit of 10.70 pg/μl DNA, was 100-fold greater than that of PCR. Thirteen infection bacterial strains without bla(NDM-1) were selected for testing of specificity, and the results of the amplification were negative, which showed that the primers had good levels of specificity. The LAMP method reported here is demonstrated to be a potentially valuable means for the detection of bla(NDM-1) and rapid clinical diagnosis, being fast, simple, and low in cost.