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.     

Discriminating between varicella-zoster virus vaccine and wild-type strains by loop-mediated isothermal amplification

The loop-mediated isothermal amplification (LAMP) method was developed to distinguish between the varicella-zoster virus (VZV) vaccine (vOka) strain and wild-type strains. Two single nucleotide polymorphisms (SNPs) (nucleotide [nt] 105705 for VR-1 VZV LAMP and nt 106262 for VR-2 VZV LAMP) located in the open reading frame 62 gene were selected as LAMP targets. Amplified vOka DNA demonstrated a typical ladder pattern; however, no LAMP product was detected in reactions performed with DNAs from other human herpesviruses by either VR-1 VZV LAMP or VR-2 VZV LAMP. This result was confirmed by a turbidity assay. The sensitivities of both VR-1 and VR-2 VZV LAMP determined by either the turbidity assay or agarose gel electrophoresis were 100 copies per reaction. To discriminate the vOka strain from wild-type strains, VR-1 and VR-2 VZV LAMP products were digested with the appropriate restriction enzymes (SacII for VR-1 LAMP and SmaI for VR-2 LAMP). The digested products were clearly different in the vOka strain and wild-type strains. To evaluate the utility of the LAMP methods for rapid differentiation, viral DNA (without DNA extraction) in swab samples was directly tested. Wild-type VZV DNA was detected in 20 swab samples by either VR-1 VZV LAMP or VR-2 VZV LAMP. Sequence analysis confirmed the expected SNPs in the LAMP products amplified from the vOka strain and the five wild-type strains.     

Detection of human herpesvirus 7 DNA by loop-mediated isothermal amplification

The reliability of loop-mediated isothermal amplification (LAMP), initially developed for the detection of human herpesvirus 7 (HHV-7), was evaluated in this study. Although a LAMP product was detected in HHV-7 DNA, neither HHV-6 nor human cytomegalovirus DNA produced a product. When agarose gel electrophoresis was used for the detection of LAMP products, the sensitivity of a 30-min HHV-7 LAMP reaction reached 250 copies/tube. The use of turbidity for the detection of the LAMP products gave a sensitivity of 500 and 250 copies/tube for 30- and 60-min reactions, respectively. Following these initial validation studies, clinical samples collected from two patients with primary HHV-7 infections were examined by HHV-7 LAMP. By use of agarose gel electrophoresis, HHV-7 LAMP products could be detected in acute-phase plasma samples but no LAMP product was detectable in convalescent-phase plasma samples from either patient. Since a turbidity assay is less sensitive than agarose gel electrophoresis, no HHV-7 LAMP product could be detected in plasma samples after a 30-min LAMP reaction. After a 60-min LAMP reaction, HHV-7 LAMP product could be detected in acute-phase plasma samples.     

Development of the loop-mediated isothermal amplification method for rapid detection of cytomegalovirus DNA

Cytomegalovirus (CMV) loop-mediated isothermal amplification (LAMP) was performed on DNA extracted from CMV (AD-169)-, herpes simplex virus (HSV) 1 (KOS)-, HSV-2 (186)-, varicella-zoster virus (Oka-vaccine)-, human herpesvirus (HHV)-6 A (U1102)-, HHV-6 B (Z29)-, and HHV-7 (RK)-infected cells. Although amplified CMV demonstrated typical ladder patterns, no LAMP product was detected in reactions performed with other viral DNAs. The sensitivity of the CMV LAMP was 500 copies/tube, as determined by either agarose gel electrophoresis or turbidity assay. To determine whether CMV LAMP could be used for quantitative analysis of viral DNA, threshold times, defined as the time (in seconds) to reach the threshold level (0.1), were measured by amplification of serial dilutions of the plasmid DNA. The standard curve exhibited a correlation coefficient of 0.944, a slope of -208.1, and a y-intercept of 3261.4. Following these initial validation experiments, we analyzed 180 samples collected serially from 20 pediatric hematopoietic stem cell transplant recipients. Detection of CMV DNA in whole blood (WB) was tested by CMV LAMP and real-time polymerase chain reaction (PCR). When >500 copies/tube (>5000 copies/200 microl of WB) was defined as positive for CMV infection, the sensitivity, specificity, positive predictive value, and negative predictive values of the CMV LAMP were 80.0, 98.9, 66.7, and 99.4%, respectively.     

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%.     

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.     

Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid technique, which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for infectious hypodermal and hematopoietic necrosis virus (IHHNV) detection was designed. A set of four primers was designed by targeting the IHHNV genome DNA. By the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by the white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. The assay had a detection limit of 5-500 copies of DNA template with gel electrophoresis, SYBR Green I and white turbidity with naked-eye inspection. The detection sensitivity of LAMP was 100-fold higher than the PCR. A diagnostic procedure which is rapid and highly sensitive was developed for IHHNV detection.     

Development of loop-mediated isothermal amplification assay for specific and rapid detection of camelpox virus in clinical samples

In this study, development of loop-mediated isothermal amplification (LAMP) assay based on ankyrin repeat protein gene (C18L) for specific and rapid detection of camelpox virus (CMLV) was carried out. The assay was optimized using viral genomic DNA (gDNA) extracted from density gradient purified CMLV and standard control recombinant DNA plasmid containing the target, which resulted in reliable amplification at 62°C for 60 min. The amplified LAMP product was identified by agarose gel electrophoresis and subsequent direct visualization under UV light or observation by naked-eye for the presence of turbidity and color change following the addition of SYBR Green I dye and hydroxy naphthol blue (HNB). The analytical specificity of LAMP and conventional PCR assays was evaluated using other related poxviruses namely buffalopox, goatpox, sheeppox, and orf viruses, which revealed only a specific amplification of CMLV. The LAMP assay was 10-fold more sensitive than the conventional PCR. Further, the assay was evaluated with DNA extracted from the cell culture isolates of CMLV (n=11) and clinical samples (n=23). These results proved that the developed LAMP is a simple, specific, sensitive, rapid and economical diagnostic tool for detection of CMLV from clinical materials.     

Loop-mediated isothermal amplification for discriminating between human herpesvirus 6 A and B

Genotyping of human herpesvirus 6 (HHV-6) is important clinically, particularly for the diagnosis of neurological diseases. The objective of this study was to establish a rapid HHV-6 genotyping method using the loop-mediated isothermal amplification (LAMP) method. An AccI site is located in the target sequence of HHV-6 B, but not in HHV-6 A. LAMP products were digested with the AccI enzyme and then separated by agarose gel electrophoresis to differentiate the digest pattern of the two variants. The fragment patterns were clearly different between HHV-6 A and B. In order to evaluate the reliability of this HHV-6 genotyping method for use in the clinical laboratory, serum samples from 20 patients with either primary HHV-6 infection or viral reactivation were collected and analyzed. HHV-6 DNA was amplified directly from the serum samples and all 20 LAMP products were positive for HHV-6 B.     

Sensitive and rapid detection of herpes simplex virus and varicella-zoster virus DNA by loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react rapidly and efficiently, with a high specificity, under isothermal conditions. We used a LAMP assay for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). The virus specificities of primers were confirmed by using 50 HSV-1, 50 HSV-2, and 8 VZV strains. The assay was performed for 45 min at 65 degrees C. The LAMP assay had a 10-fold higher sensitivity than a PCR assay. An analysis of nucleotide sequence variations in the target and primer regions used for the LAMP assay indicated that 3 of 50 HSV-1 strains had single nucleotide polymorphisms. No HSV-2 or VZV strains had nucleotide polymorphisms. Regardless of the sequence variation, there were no differences in sensitivity with the HSV-1-specific LAMP assay. To evaluate the application of the LAMP assay for clinical diagnosis, we tested clinical samples from 40 genital herpes patients and 20 ocular herpes patients. With the LAMP assay, 41 samples with DNA extraction and 26 direct samples without DNA extraction were identified as positive for HSV-1 or HSV-2, although 37 samples with DNA extraction and just one without DNA extraction were positive by a PCR assay. Thus, the LAMP assay was less influenced than the PCR assay by the presence of inhibitory substances in clinical samples. These observations indicate that the LAMP assay is very useful for the diagnosis of HSV-1, HSV-2, and VZV infections.     

A rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2

BackgroundA simple and rapid IsoAmp^® HSV assay has been developed for qualitative detection of herpes simplex virus (HSV) types 1 and 2 from genital lesions. Sample preparation involved a simple dilution step and the diluted specimens were directly added to the device and amplified by isothermal helicase-dependent amplification (HDA). Amplification products were then detected by a DNA strip embedded in a disposable cassette without any instrument. The total test turn-around time is less than 1.5 h from specimen processing to result reporting.ObjectivesTo evaluate the analytical and clinical performance of the IsoAmp^® HSV assay as well as the robustness and reproducibility of the assay.Study designThe analytical sensitivity of the IsoAmp^® HSV assay was determined using both HSV-1 and HSV-2. Clinical performance was evaluated using 135 frozen specimens collected from patients with suspected HSV infection in genital area.ResultsThe analytical sensitivity of the assays was 5.5 and 34.1 copies/reaction for HSV-1 and HSV-2 respectively with a 95% confidence interval. When the herpes viral culture was used as the reference standard, the clinical sensitivity and specificity of the IsoAmp^® HSV assay were 100.0% and 96.3% respectively. The inter-laboratory reproducibility achieved an overall 97.5% agreement by testing a total of 80 blinded HSV-1 samples among five laboratories.ConclusionAdequate analytical and clinical performance of the IsoAmp^® HSV assay was demonstrated. This assay is simple to perform and has acceptable inter-laboratory reproducibility.     

Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections

A novel multiplex nested polymerase chain reaction (PCR) assay was designed and evaluated for routine diagnosis of herpes simplex virus (HSV) infections in patients with either putative HSV infection of the central nervous system or suspected HSV keratitis. Single-tube amplification of HSV type 1 (HSV-1) or type 2 (HSV-2) DNA extracted from cerebrospinal fluid (CSF) or from keratectomy specimens was followed by differentiation of the virus type-specific PCR products either by agarose gel analysis or by DNA enzyme immunoassay. Among 417 CSF specimens obtained from 395 consecutive patients with clinically suspected HSV infection, 11 (2.6%) were positive for HSV-1 DNA and four (1.0%) probes were positive for HSV-2 DNA. None of the specimens was positive for both HSV-1 and HSV-2 DNA. The genome of HSV-2 was detected in a CSF sample obtained from a woman with meningoencephalitis and genital herpes. The presence of PCR inhibitors was detected in six of 111 (5.4%) reconstructed CSF samples. Inhibition could be removed following extraction with a commercial kit. HSV-1 DNA, but no HSV-2 DNA, was detected in corneal buttons obtained from patients with suspected herpetic keratitis. No contamination has been recorded during the 2-year routine use of this test, which has met the specific requirements of a diagnostic laboratory. © 1996 Wiley-Liss, Inc.     

Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 10(8) copies of HSV DNA/20 microl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.     

Loop-mediated isothermal amplification establishment for detection of pseudorabies virus

A rapid, convenient and reliable pseudorabies virus (PRV) detection system was developed by using the loop-mediated isothermal amplification (LAMP) method. Six special primers were designed successfully based on the PRV DNA-binding protein (DBP) gene. The assay was optimized to amplify PRV DNA by incubation at 63 degrees C for 1h. The LAMP products had a ladder-like pattern of bands from 188 bp when electrophoresed on an agarose gel and its specificity was confirmed by digestion with Hinc II enzyme. Two naked-eye detection methods were developed for use in the field. The detection limit of the LAMP assay was found to be 10 fg DNA sample which was 100-1,000-fold higher than that of PCR. By using DNA (or cDNA) samples extracted from three different PRV strains and six other viruses known to be related genetically to PRV or to cause similar clinical signals in pig, the system was identified to amplify only the PRV DNA. A comparison between the LAMP and PCR assay using five clinical samples showed good correlation.     

环介导等温扩增技术检测卡氏肺孢子虫的研究

目的 环介导等温扩增(LAMP)技术检测卡氏肺孢子虫(Pc).方法 醋酸可的松经皮下注射Wistar大鼠诱导Pc,收集支气管肺泡灌洗液(BALF)提取Pc基因组DNA.设计4条扩增Pc线粒体核糖体大亚基(mtrRNA)基因的LAMP引物,以结核杆菌、肺炎支原体、肺炎衣原体、弓形虫、大鼠白细胞为对照,进行LAMP反应.LAMP产物经显色、电泳及酶切鉴定.将Pc DNA 10倍稀释后同时进行LAMP和PCR,比较其敏感性.结果 Pc检测管经显色后呈绿色(阳性),对照组均呈棕色(阴性).Pc LAMP产物经电泳后呈LAMP特征性梯状条带,扩增产物经Tail限制性内切酶酶切鉴定正确,对照组均无扩增产物.LAMP可检测到虫体DNA的最低浓度是lP9/pJ,为PCR的10倍.结论 检测Pc的LAMP方法敏感、特异及简便.     

Variations in the cytomegalovirus major immediate-early gene found by direct genomic sequencing.

An assay to detect and sequence DNA from human cytomegalovirus (HCMV) immediate-early gene region 1 has been developed; it involves in vitro amplification by the polymerase chain reaction and direct solid-phase sequencing of the amplified material. Urine samples from 16 patients tested positive for HCMV DNA in both a colorimetric assay for the detection of immobilized amplified nucleic acids and a standard polymerase chain reaction assay with agarose gel electrophoresis. Ten urine samples from healthy people tested negative in the same assays. Analysis of 106-bp fragments from seven patients and two laboratory HCMV strains (Ad 169 and Towne) demonstrated that the viral sequences were conserved in samples collected at different times from the same patient and in tissue-cultured samples. Two of the patient strains had variations in the amplified region, with a total of seven nucleotide substitutions yielding five amino acid alterations in the coding sequence.     

Simultaneous detection of herpes simplex virus types 1 and 2 by real-time PCR and Pyrosequencing.

BACKGROUND: Up to 80% of the US adult population has been exposed to herpes simplex virus (HSV) type 1, primarily during childhood. Also, approximately 20% of the US population has contracted genital herpes from HSV-2 infections. Clinical symptoms can present as fever, headache, malaise, myalgia, and cold sores/lesions that cause pain, itching, dysuria, and vaginal or urethral discharge. A recurrence of infection is common. HSV culturing is characterized by low sensitivity with variable success rates due to shipping conditions. OBJECTIVE: To design and validate a real-time PCR assay capable of simultaneously detecting each HSV subtype. STUDY DESIGN: ATCC-purchased HSV-1 and HSV-2 positive samples and HSV-1 and HSV-2 infected clinical specimens were assayed simultaneously with shared amplification primers and subtype-specific probes against the HSV glycoprotein B gene on a Rotor-Gene 3000 platform. Separately, two PCR reactions were performed in which one primer contained a 5' biotin modification. Single-stranded DNA from the amplicon was purified and Pyrosequenced. RESULTS: The quantitative range of the assay extended from 10(8) through 10(0) copies of each virus (r(2) > 0.991) and specificity was determined by non-amplification of 37 different human pathogens, including other herpesviruses such as VZV, CMV, and EBV. Sensitivity and specificity values of 100% were calculated by concordance analysis between the real-time PCR and the DNA Pyrosequencing results (HSV-1: n = 119, HSV-2: n = 120). Application of this assay to 4581 cervical swab specimens collected from women visiting physicians primarily in six states provided detection rates of 3.1% for HSV-1 and 7.6% for HSV-2. The average age of women infected with HSV-1 was 29.5 versus 35.6 for HSV-2. CONCLUSIONS: This procedure was demonstrated as both highly sensitive and specific for the detection of HSV-1 and HSV-2 in a single reaction. Also, the integration of Pyrosequencing analysis permitted an innovative and rapid verification for each subtype.     

Rapid detection of human herpesvirus 8 DNA using loop-mediated isothermal amplification

The reliability of a loop-mediated isothermal amplification (LAMP) method for the detection of human herpesvirus 8 (HHV-8) DNA was evaluated. Although LAMP products were produced with the DNA sample extracted from BCP-1 cells, LAMP products were not produced with the DNAs from seven other human herpesviruses. The detection limit of the HHV-8 LAMP method was 100 copies of target sequence/tube. To determine whether the HHV-8 LAMP method could be used to quantify viral DNA, threshold times, which are defined as the time (in s) it takes to reach the threshold turbidity level (0.1), were measured for the amplification of serial dilutions of a DNA plasmid containing the target sequence. The standard curve possessed a correlation coefficient of 0.9428 with a slope of -84.079 and y-intercept value of 1936.2. Additionally, an attempt was made to detect viral DNA in 17 specimens collected from Kaposi's sarcomas and two cell lines obtained from primary effusion lymphomas. HHV-8 DNA was detected in 14 of the 17 Kaposi's sarcoma tissue samples and both of the primary effusion lymphoma cell lines. Viral DNA was not detected in HHV-8 LAMP-negative samples using the real-time PCR method.     

Rapid detection of the most common high-risk human papillomaviruses by loop-mediated isothermal amplification

Persistent infection with high-risk human papillomavirus (HPV) is a major risk factor for development of cervical cancer. At present, polymerase chain reaction (PCR)-based methods, the most widely molecular tools used for HPV detection, are time-consuming and require expensive instruments. In this study, loop-mediated isothermal amplification (LAMP) was established for detection of HPV types 16, 18, 45 and 58 which are frequently found in Thailand. The optimal condition for detection of these high risk HPVs was 63°C for 60min. Since a white magnesium pyrophosphate precipitate is a characteristic by product of the LAMP reaction which can be visualized directly by the naked eye, the entire assay time of LAMP is 1h compared to 6-8h of for a nested PCR detection. The detection limit of LAMP assay was shown to be equivalent to nested PCR that could amplify 10(2) copies of HPV-18 and 10(3) copies of HPV 16, 45 and 58, as determined by either turbidity detection or agarose gel electrophoresis. No cross-reaction was observed, indicating that LAMP assay has high type-specificity. The assay showed successful detection of HPV in 56 clinical specimens. Using nested PCR as the gold standard, the sensitivity, specificity, negative predictive values and positive predictive values of LAMP assay were 100%. In conclusion, LAMP assay is a high efficiency, low cost diagnostic tool, useful for rapid, accurate, direct detection of HPV for clinical diagnosis.     

Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65 °C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis.