Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.     

Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification.

A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river water were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.     

Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae . Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (χ² test, P <0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Design and Development of a Multiplex Real-Time PCR Assay for Detection of HIV-1 and HCV Using Molecular Beacons

At least 10 million individuals worldwide are co-infected with immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). These two viruses are transmitted most primarily by exposure to infected blood or blood products. Various nucleic acid assays have been developed for diagnostics and therapeutic monitoring of infections. In the present study, a multiplex real-time PCR assay for simultaneous detection of HCV and HIV-1 using molecular beacons were designed and validated. A well-conserved region in the HIV-1 pol gene and 5′NCR of HCV genome were used for primers and molecular beacon design. The analysis of scalar concentrations of the samples indicated that this multiplex procedure detects at least 1,000 copies/ml of HIV-1 and 100 copies/ml of HCV with linear reference curve (R ² > 0.94). The results demonstrate that a specificity of 100 % and sensitivity of 96 % can be achieved. The analytical sensitivity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes only detected HIV-1 and all major variants of HCV. This assay may represent an alternative rapid and relatively inexpensive screening method for detection of HIV-1/HCV co-infection especially in blood screening.     

Evaluation of the usefulness of various PCR method variations and nucleic acid hybridization for CMV infection in immunosuppressed patients

In diagnosis of CMV infection various laboratory methods are used. The methods based on detection of viral nucleic acids have been introduced routinely in many laboratories. The aim of this study was to compare nucleic acid hybridisation method and various variants of PCR methods with respect to their ability to detect CMV DNA. The studied material comprised 60 blood samples from 19 patients including 13 renal transplant recipients and 6 with acute leukaemia. The samples were subjected to hybridisation (Murex Hybrid Capture System CMV DNA) and PCR carried out in 3 variants: with one pair of primers (single PCR), nested PCR and Digene SHARP System with detection of PCR product using a genetic probe in ELISA system. The sensitivity of the variants ranged from 10(0) particles of viral DNA in nested PCR to 10(2) in single PCR. The producer claimed the sensitivity of the hybridisation test to be 3 x 10(5) and it seems to be sufficient for detection of CMV infection. The obtained results show that sensitivity of hybridisation was comparable to that of single PCR and the possibility of obtaining quantitative results makes it superior, on efficacy of antiviral therapy, especially in monitoring CMV infection in immunossuppressed patients and in following the efficacy of antiviral treatment.     

弓形虫（Toxoplasma gondii）的PCR检测方法在笼养猕猴群中的应用

目的建立快速、灵敏、特异及检测结果易判断的PCR方法,并应用于大规模猕猴种群的弓形虫常规检测中。同时比较巢式PCR和单一PCR的一致性。方法根据弓形虫保守基因p30（SAG1）设计了内、外两对进行巢式PCR扩增以及B1基因设计一对引物进行单一PCR扩增,将DNA样本进行10倍倍比稀释,以检测巢式PCR反应的灵敏度;并对医学生物学研究所自繁猕猴共150只进行了弓形虫检测。结果巢式PCR检测法检测限度可达10^-3ng/uL,而且方法特异。两种PCR法检测结果基本一致,其中巢式PCR检测阳性率（10％）稍高于单一PCR检测阳性率（8．67％）。结论巢式PCR和一次PCR方法都可应用于猕猴弓形虫的常规检测中,并提示巢式PCR比单一PCR更敏感、检出率更高。     

Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.     

Detection of low numbers of Salmonella in environmental water, sewage and food samples by a nested polymerase chain reaction assay

A polymerase chain reaction (PCR) assay with two nested pairs of primers selected from conserved sequences within a 2.3 kb randomly cloned DNA fragment from the Salmonella typhimurium chromosome was developed. The nested PCR assay correctly identified 128 of a total of 129 Salmonella strains belonging to subspecies I, II, IIIb and IV. One strain of Salm. arizona (ssp. IIIa) tested negative. No PCR products were obtained from any of the 31 non-Salmonella strains examined. The sensitivity of the assay was 2 cfu, as determined by analysis of proteinase K-treated boiled lysates of Salm. typhimurium. The performance of the assay was evaluated for environmental water, sewage and food samples spiked with Salm. typhimurium. Water and sewage samples were filtered and filters were enriched overnight in a non-selective medium. Prior to PCR, the broth cultures were subjected to a rapid and simple preparation procedure consisting of centrifugation, proteinase K treatment and boiling. This assay enabled detection of 10 cfu 100 ml(-1) water with background levels of up to 8700 heterotrophic organisms ml(-1) and 10000 cfu of coliform organisms 100 ml(-1) water. Spiked food samples were analysed with and without overnight enrichment in a non-selective medium using the same assay as above. Nested PCR performed on enriched broths enabled detection of <10 cfu g(-1) food. Variable results were obtained for food samples examined without prior enrichment and most results were negative. This rapid and simple assay provides a sensitive and specific means of screening drinking water or environmental water samples, as well as food samples, for the presence of Salmonella spp.     

Inhibition of Legionella pneumophila PCR in respiratory samples: A quantitative approach

Impurities in complex biological samples that persist through nucleic acid preparation can inhibit PCR or reduce the sensitivity and efficiency of PCR amplification and thus affect reliable results in PCR diagnostics. To obtain information on the incidence of inhibition events and on the magnitude of the loss of sensitivity, respectively, we devised a relative inhibition assay and examined 100 samples from patients with respiratory tract infections. As a reference, samples were spiked with Legionella (L.) pneumophila. Detection was by standard nucleic acid purification and subsequent real-time PCR. By comparing the crossing points of the fluorescent curves to those of an L. pneumophila standard dilution series, we were able to quantify the respective degrees of inhibition into several categories. We found complete inhibition in 2% of the samples. 12% were not reliably detected. 65% of the tested samples showed moderate to strong inhibition, but were still reliably detected, whereas in 21% of the samples no inhibition was observed. Except for a significantly higher inhibition in tracheal aspirates than in BAL samples, the degree of inhibition did not correlate with the physical properties of the respective sample. The relative inhibition assay established an unexpectedly broad distribution of the inhibition-degrees in inflammatory respiratory materials.     

Evaluation of the Bio-Rad Geenius HIV 1/2 Confirmation Assay as an Alternative to Western Blot in the Korean Population: A Multi-Center Study

Recently updated recommendations for diagnosis of HIV infection suggest a new diagnostic algorithm including HIV-1/HIV-2 antibody differentiation immunoassay instead of western blot (WB) as a confirmatory testing. We evaluated Bio-Rad Geenius HIV1/2 confirmation assay as a simple and reliable alternative to WB in the Korean population with low HIV prevalence. The Geenius HIV1/2 was performed in a total of 192 serum specimens (140 reactive and 52 nonreactive specimens by ARCHITECT HIV Ag/Ab Combo assay) that were prospectively collected from five institutions. HIV-1 nucleic acid amplification test (NAT) was performed in negative or indeterminate specimens by Geenius HIV1/2 or WB. Among 140 reactive specimens by HIV Ag/Ab assay, 82 (58.6%) were positive for HIV-1 Ab by Geenius HIV1/2. Among 58 negative or indeterminate specimens by Geenius HIV1/2, four specimens (6.9%) were positive by HIV-1 NAT. The sensitivity and specificity of Geenius HIV1/2 were 95.3% and 100.0%, respectively. When we considered only WB, the sensitivity and specificity of Geenius HIV1/2 were 100.0% and 99.1%, respectively. Agreement between Geenius HIV1/2 and WB was excellent (weighted Kappa = 0.89). The Geenius HIV1/2 is simple and time-saving compared with WB. It has an excellent performance and can be a reliable alternative to WB. HIV-1 NAT should be performed in negative or indeterminate specimens by Geenius HIV1/2 to detect acute HIV infection as recommended in new HIV testing algorithms.     

丙型肝炎病毒RNA打点杂交检测方法同RT－PCR方法的比较

采用ＨＣＶ基因组结构区Ｃ区ｃＤＮＡ探针和非结构区ＮＳ３－４区ｃＤＮＡ探针,建立了用打点杂交（ｄｏｔｂｌｏｔｈｙｂｒｉｄｉｚａｔｉｏｎ）检测血清中ＨＣＶＲＮＡ的方法,同采用ＨＣＶ基因组５’端非编码区的一对寡核苷酸引物通过逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）检测血清中ＨＣＶＲＮＡ的方法相比较,发现两种方法都能快速早期和特异地检出血清中ＨＣＶＲＮＡ,但ＲＴ－ＰＣＲ法敏感性优于ＲＮＡ打点杂交法。对于无血清学指标的慢性ＮＡＮＢ肝炎病人的诊断,可采用这两种方法。这两种方法的敏感性在很大程度上依赖于引物和探针的敏感性,以及ＲＮＡ提取方法。ＲＴ－ＰＣＲ法适用于诊断病毒血症和复制,打点杂交法适用于研究ＨＣＶＲＮＡ量的变化,对治疗的评价,以及为实验筛选较高滴度的ＨＣＶＲＮＡ阳性样本。     

Detection of mouse parvovirus in Mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay

We used primary and nested polymerase chain reaction (PCR) assays to determine the presence of mouse parvovirus (MPV) in mouse sperm, oocytes, preimplantation embryos, and ovarian tissues collected from MPV-infected mice. The primary PCR assay detected MPV in 56% of the sperm samples. MPV was not eliminated by passing sperm samples through a Percoll gradient. After Percoll treatment, MPV was still present in 50% of the samples according to primary PCR assay. Oocyte samples that did not undergo extensive washing procedures had detectable MPV in 7% of the samples based on the primary PCR assay, but nested PCR assay detected higher (28%) infection rate. However, MPV was not detected in oocytes that underwent extensive washing procedures, as assessed by either primary or nested PCR assay. Although primary PCR did not detect MPV in embryos, a nested PCR assay determined that 50% of the embryos were positive for the virus. In addition, ovarian tissues were collected from 3 different mouse colonies with enzootic MPV infection. Ovarian tissue collected from 129CT, 101/R1, and Sencar mice had high incidence (38%, 63%, and 65%, respectively) of MPV infection on the basis of nested PCR amplification. These results demonstrate that mouse gametes, embryos, and ovarian tissues may be contaminated with MPV and therefore caution is necessary when infected germplasm is used for assisted reproductive technologies such as embryo transfer, establishing embryonic stem cell lines, in vitro fertilization, ovary transplantation, and intracytoplasmic sperm injection.     

A sensitive nonisotopic hybridization assay for HIV-1 DNA

We have developed a microtiter-based sandwich hybridization assay for the detection of low copy number HIV-1 sequences. The assay employs a capture DNA sequence covalently coupled to microtiter wells through linker arms. The detection probe is a biotin-labeled DNA fragment derived from sequences adjacent to the capture sequence. After hybridization in the presence of sample nucleic acid, the detection probe remains bound only if the sample contained complementary sequences spanning the junction between capture and detection probes. The amount of detection probe bound is quantified by incubation with a peroxidase-streptavidin conjugate and a colorimetric peroxidase substrate. This assay has been combined with enzymatic target amplification to achieve sensitive detection of HIV-1 in patient samples. Following amplification of HIV-1 DNA using the polymerase chain reaction technique, a 190-bp product is produced. This product is easily and specifically quantified using the sandwich hybridization assay. The resulting test can detect one HIV-1-infected cell in 10(5) cells or about 30 molecules of HIV-1 DNA.