Conventional Nested Polymerase Chain Reaction Research Articles

Genetic diversity of Chlamydia among captive birds from central Argentina

To study the occurrence of Chlamydia spp. and their genetic diversity, we analysed 793 cloacal swabs from 12 avian orders, including 76 genera, obtained from 80 species of asymptomatic wild and captive birds that were examined with conventional nested polymerase chain reaction and quantitative polymerase chain reaction. Chlamydia spp. were not detected in wild birds; however, four species (Chlamydia psittaci, Chlamydia pecorum, Chlamydia pneumoniae and Chlamydia gallinacea) were identified among captive birds (Passeriformes, n = 20; Psittaciformes, n = 15; Rheiformes, n = 8; Falconiformes n = 2; Piciformes n = 2; Anseriformes n = 1; Galliformes n = 1; Strigiformes n = 1). Two pathogens (C. pneumoniae and C. pecorum) were identified simultaneously in samples obtained from captive birds. Based on nucleotide-sequence variations of the ompA gene, three C. psittaci-positive samples detected were grouped into a cluster with the genotype WC derived from mammalian hosts. A single positive sample was phylogenetically related to a new strain of C. gallinacea. This report contributes to our increasing understanding of the abundance of Chlamydia in the animal kingdom.

Experimental Toxoplasma gondii oocyst infections in turkeys (Meleagris gallopavo)

Toxoplasma (T.) gondii is a protozoan parasite with a broad range of intermediate hosts. Humans are often infected by ingestion of tissue cysts in raw or undercooked meat or meat products. Turkeys as food-producing animals can also serve as intermediate hosts. The aim of the present study was to investigate occurrence and predilection sites of T. gondii infection in turkeys after oral infection with oocysts. Experimental infections with different doses of T. gondii oocysts were performed in 36 turkeys to mimic natural infection. Systemic distribution of parasitic stages was investigated by screening 14 different tissues including the edible tissues heart, liver, thigh, breast and drumstick muscle. Parasite detection was based on a conventional nested polymerase chain reaction (PCR). Animals were sacrificed 6–12 weeks after infection. Results demonstrated parasite spreading over the whole organism after oral infection by oocysts. Most frequently affected tissues were brain (47.2% of all brains were positive for T. gondii) and thigh muscle (25.0% positive samples). Other muscles were regularly T. gondii-positive, all other sampled tissues were positive at least once. Thus, edible tissues are one of the predilection sites of T. gondii in turkeys which renders raw or undercooked turkey meat a potential risk for parasite transmission to humans. Data were compared to results from previous parenteral turkey infections with tachyzoites. With the exception of brain, liver and breast muscle affection, no significant differences were observed between both infection routes. Both infection models could be used for research purposes with certain advantages and disadvantages.

Comparative Analysis of Polymerase Chain Reaction Assay for Herpes Simplex Virus 1 Detection in Tear

PurposeTo comparatively analyze the methodological efficacy of the polymerase chain reaction (PCR) assay for herpes simplex virus 1 (HSV) detection in tears.MethodsThis retrospective study reviewed the medical records of 115 patients who were clinically diagnosed with herpes keratitis, and their tear samples were collected for HSV detection. PCR positive rates were analyzed for their dependence on the PCR primers used (conventional PCR primer vs. nested PCR primer), the tear collecting method used (micropipetting vs. collection with schirmer strip), the disease manifestation and the patient's previous medication history.ResultsHSV DNA was detected in 23 out of 115 (20%) tear samples. The PCR positive rate in tear samples did not differ depending on the PCR primer or tear collection method used. Typical epithelial lesions showed a higher positive rate (31.4%) than atypical epithelial lesions (10.9%). The previous history of the antiviral agent seemed to affect the PCR positive rate.ConclusionsAlthough the PCR positive rate was not dependent on the tear collection method or primers, HSV detection in tears using PCR was shown to be a supplementary diagnostic test in typical and atypical herpes epithelitis.

Assessment of three commercial DNA extraction kits and a laboratory-developed method for detecting Cryptosporidium and Cyclospora in raspberry wash, basil wash and pesto

Polymerase chain reaction (PCR) methods are often used to identify the parasitic protozoa Cryptosporidium parvum and Cyclospora cayetanensis in foods although little has been published regarding the efficacy of available DNA extraction methods. This study reviewed three commonly used commercial DNA extraction kits: FastDNA SPIN Kit for soil, QBiogene (FastDNA), UltraClean™ Soil DNA Isolation Kit, MO BIO Laboratories (MoBio), and QIAamp DNA Mini Stool Kit, Qiagen (QIAamp), as well as a ‘homebrew’ Universal Nucleic Acid Extraction (UNEX) method.Washes from raspberry and basil as well as commercial pesto samples were seeded with 5000, 500, or 50 C. parvum and C. cayetanensis oocysts. The protocols were assessed for: quantity and quality of the extracted DNA, time to completion, presence of PCR inhibitors and the percentage of samples correctly identified as positive for the two parasites.Real-time and conventional nested PCR assays were used to detect the seeded pathogens. Of the commercial kits, PCR results of samples extracted using FastDNA were statistically similar to QIAamp and both were superior to MoBio. Differences in PCR results among FastDNA, QIAamp and UNEX for detection of Cyclospora were not statistically significant although the UNEX method proved best with Cryptosporidium. Real-time PCR assays targeted the 18S rRNA and the hsp70 genes of C. cayetanensis; overall results were similar to those found using conventional nested PCR targeting the 18S rRNA gene.

Multiplex high resolution melting assay for estimation of Porcine Endogenous Retrovirus (PERV) relative gene dosage in pigs and detection of PERV infection in xenograft recipients

Porcine Endogenous Retrovirus (PERV) poses an infectious risk in the field of xenotransplantation. This risk may be mitigated by breeding selectively animals bearing favorable PERV genetic characteristics including pigs with low levels of PERV integrated in the genome. A real-time quantitative polymerase chain reaction (PCR) assay employing the Roche High Resolution Melting (HRM) Master was used to estimate the relative gene dosage of PERV pol integrated within the pig genome. When assessed across 99 pigs of the Auckland Island breed numerous animals bearing low gene dosage were identified. The assay was adapted further to perform multiplex PCR for the detection of PERV infection within xenograft recipients. Besides PERV, amplification targets for the multiplex PCR include a pig cell marker for the determination of microchimerism and an internal amplification control (IAC) to assess the efficiency of nucleic acid isolation and effects of PCR inhibition. When 12 patients who had received porcine islet transplants were tested no evidence of PERV infection was found. The assay was shown to be specific, highly reproducible with superior performance over conventional nested PCR. This assay can be used as both a screening tool for PERV proviral levels within donor pigs and as a diagnostic tool to examine PERV transmission in human patients treated with porcine xenotransplantation material.

An improved Real-Time Polymerase Chain Reaction for the Simultaneous Detection of All Serotypes of Epizootic Hemorrhagic Disease Virus

Epizootic hemorrhagic disease virus (EHDV) is a significant pathogen of wild and sometimes domestic ungulates worldwide. Rapid and reliable methods for virus detection and identification play an essential part in the control of epizootic hemorrhagic disease (EHD). In the present study, a 1-step real-time polymerase chain reaction (PCR) group-specific assay was developed. The assay detects genome segment 5 (NS1) from all of the 8 serotypes of EHDV. Assay sensitivity was evaluated relative to a conventional gel-based nested PCR using cell culture-derived virus and diagnostic samples from clinically affected white-tailed deer (Odocoileus virginianus). The assay reliably amplified the NS1 gene from any of the EHDV strains tested, including isolates from each of the 8 EHDV serotypes. No cross-reactions were detected when all 24 serotypes of Bluetongue virus, a closely related member of the genus Orbivirus, were tested. A panel of 76 known EHDV-positive clinical samples was used to compare the performance of the assay relative to a previously reported real-time PCR assay. Results indicated that there was no statistically significant difference between the threshold cycle values obtained with both assays. A collection of 178 diagnostic samples submitted for EHD diagnosis was also used for test evaluation. The assay could be applied for rapid detection of EHDV in clinical samples from susceptible ruminants during an outbreak of the disease. In addition, this PCR assay has the benefits of being reliable and simple and could provide a valuable tool for studying the epidemiology of EHDV infection in susceptible ruminants by facilitating the detection of EHDV, regardless of the serotype.

A Simplified Real-Time PCR Method for the Quantification of Minimal Residual Disease in Follicular Lymphoma.

High-dose therapy and autologous stem-cell transplantation (HDT/ASCT) is associated with long remissions in relapsed follicular lymphoma (FL). Molecular remission in the graft and post ASCT is predictive of durability of remission. Thus, a simple, reliable method of quantitation of minimal residual disease (MRD) would improve prognostication in these patients. Real-time polymerase chain reaction (PCR) can quantitatively detect FL markers in blood and BM with a reproducible sensitivity of 0.01%. Fluorescent probe systems are used to perform real-time PCR; however, these techniques are expensive and do not allow melt curve analysis of the PCR product. We describe here a less expensive SYBR green detection method which also enables us to check clonal identity, and yields sensitive, reliable quantitative results for monitoring MRD. We compared our real-time PCR results with previously determined conventional nested PCR results in a series of samples collected from patients enrolled in one of our clinical trials.Methods: Patients with relapsed FL (n=14) were enrolled in a prospective study of HDT/ASCT followed by maintenance Interferon-a 3x106 U/m2 SC 3 x weekly for 2 yrs. DNA extracts were collected at enrolment from samples of lymph node, BM and blood to determine detection of BCL2/JH translocation. Stem-cell graft DNA and serial post-ASCT blood and BM DNA samples were collected. BCL2/JH was qualitatively determined by nested PCR using standard primers and analyzed by gel electrophoresis. For patients in whom BCL2/JH was not detectable, clone-specific IgH region primers were used where possible. Quantitative PCR was performed using QuantiTect SYBR Green kits. Melt curve analysis was performed to confirm amplification specificity and clonal identity of each sample. All samples were analyzed in triplicate for accuracy.Results: At a median follow-up of 75 months (9–91), 10/14 patients are alive. Of these, 3 are free from progressive disease. Median progression-free survival is 42 months; median overall survival has not been reached. We were successful in defining molecular markers for 12/14 patients. In 11/12, we were able to PCR amplify the BCL2/JH translocation. In one patient, allele-specific clonal IgH was analyzed with patient specific oligonucleotides. Median BCL2/JH contamination of stem-cell grafts was 0.1% (range 0.01% – 13%). 6 patients who had measurable graft contamination became molecularly negative in blood and BM within 12 months after ASCT. Increasing fractions of BCL2/JH positive blood and BM cells reliably predicted morphologic and clinical relapse. Correspondingly, the 3 patients free of disease progression have sustained very low levels of molecular evidence of FL. In one illustrative case, a patient had a long-term low-level persistence of tumour cells in BM and blood with no clinical symptoms for 70 months post-ASCT. At 76 months there was a 10-fold increase in the quantity of PCR product in both BM and blood. Radiological evidence of relapse was observed 3 months later. The patient underwent spontaneous disease regression, which was paralleled by a 10-fold decrease in PCR product quantity at 82 months.Conclusions: Our simplified method of detection using quantitative PCR yields reliable measurements of graft contamination and MRD post ASCT, which may be useful in predicting patient relapse.

The Use Of Real-Time Quantitative Polymerase Chain Reaction To Detect Hypermethylation Of The Cpg Islands In The Promoter Region Flanking The Gstp1 Gene To Diagnose Prostate Carcinoma

We developed a real-time, quantitative, methylation sensitive polymerase chain reaction (PCR) protocol to analyze hypermethylation of the CpG islands in the promoter region of the pi class glutathione-S-transferase gene GSTP1 in prostate cancer tissue. A total of 21 prostate cancer and 72 benign prostate hyperplasia (BPH) tissue samples were analyzed. Genomic DNA was digested with restriction enzyme, followed by real-time quantitative PCR amplification. Cycle threshold values were used to determine whether cancer genome was present in these tissues. A cutoff cycle threshold value of 35 was arbitrarily assigned. Samples with a cycle threshold of 35 or less were considered positive for prostate cancer. Conventional nested PCR was also performed for comparison. The mean cycle threshold values plus or minus standard deviation in prostate cancer and BPH cases were 30.12 +/- 2.88 and 37.77 +/- 2.72, respectively. All prostate cancer samples analyzed showed positive results, while 5 of the 72 BPH samples tested positive. Conventional nested PCR data indicated that 19 of 21 prostate cancer cases were positive for the methylation change, while 71 of 72 BPH cases tested negative. The test limitations of real-time PCR and the nested PCR protocols were determined to be 0.048 and 0.64 ng. DNA, respectively. We established a novel protocol for detecting the methylation change in the 5' regulatory sequence flanking the GSTP1 gene. The sensitivity of this protocol was superior to that of conventional nested PCR. The data also suggest that this novel protocol may accurately discriminate prostate carcinoma from BPH.

Differentiation of porcine circovirus (PCV)-1 and PCV-2 in boar semen using a multiplex nested polymerase chain reaction

A multiplex nested polymerase chain reaction (PCR) was developed for the detection of and differentiation between porcine circovirus (PCV)-1 and PCV-2 in boar semen. Eighteen (30%) and 30 (50%) out of 60 whole semen samples were found to be positive for PCV using multiplex conventional PCR and multiplex nested PCR, respectively. Of the 30 positive samples obtained using multiplex nested PCR, two were found to be positive for PCV-1 only, eight for PCV-2 only, and 20 for PCV-1 and PCV-2. When the separated fractions of PCV-contaminated semen were analyzed using multiplex nested PCR, PCV DNA was found to be present mainly in the seminal fluid and nonsperm cell fractions. When compared with the virus isolation method commonly used to detect viruses, this PCR assay was found to be more sensitive and rapid and, as such, may prove to be a good alternative method for the detection of and differentiation between PCV-1 and PCV-2 in boar semen.

Comparative study of conventional and novel strategies for the detection of hepatitis C virus RNA in serum: amplicor, branched-DNA, NASBA and in-house PCR

The aim of this study was to compare the sensitivity and specificity of conventional procedures (in-house one-stage polymerase chain reaction (PCR) and in-house nested PCR) and of new technologies (rTth DNA polymerase (Amplicor), branched-DNA, NASBA (nucleic acid amplification system)) for the qualitative detection of hepatitis C virus (HCV) RNA in serum of HCV-infected individuals. Serum samples from 37 anti-HCV-positive individuals (15 with a normal alanine aminotransferase (ALT) level, 22 with an elevated ALT level) and 10 anti-HCV-negative individuals as negative controls were studied. A second panel, including 9 diluted serum samples (from 1/10 to 1/100,000) was constituted to establish the differences of sensitivity of the 5 procedures with small quantities of HCV RNA in the serum. The anti-HCV-positive individuals with elevated ALT gave positive results with all 5 procedures. In patients with a normal ALT level, the assays with the highest sensitivity were Amplicor, NASBA and nested RT-PCR, followed by one-stage RT-PCR, then branched-DNA. One false-positive result was observed with Amplicor, and two with in-house nested PCR. On diluted samples, Amplicor, NASBA and nested PCR appeared more sensitive than one-stage PCR and branched-DNA. It is concluded that new procedures have satisfactory sensitivity and specificity and could advantageously replace the conventional PCR procedures for the routine qualitative detection of serum HCV RNA.

Hepatitis C viremia in chronic liver disease: Relationship to interferon-α or corticosteroid treatment

We assessed the pattern of hepatitis C viremia in chronic liver disease by studying 100 hepatitis C virus antibody-positive patients: 48 with chronic hepatitis, 21 with cirrhosis and 31 with hepatocellular carcinoma and cirrhosis. Serum hepatitis C virus RNA was detected by means of both the conventional nested polymerase chain reaction and a newly developed assay based on branched DNA that can also quantify viremia. Hepatitis C virus RNA was found in 94 of 100 patients with polymerase chain reaction and in 71 of 100 patients with branched-DNA (p < 0.001). Mean viremia level (x 10(3) genome equivalents/ml +/- S.D.), as assessed with the branched-DNA test, was 5,700 +/- 7,618 in the 48 patients with chronic hepatitis, 3,340 +/- 3,633 in the 21 patients with cirrhosis and 1,768 +/- 2,770 in the 31 patients with hepatocellular carcinoma (p < 0.02). We also analyzed retrospectively the relationship between viremia and treatment. Fifty-five patients (41 chronic hepatitis, 14 cirrhosis) underwent interferon-alpha treatment. Mean viremia level was comparable among the 30 responders (5,644 +/- 8,207) and the 25 nonresponders (5,519 +/- 6,208) to interferon, but it was significantly lower (1,841 +/- 1,864) in the 12 of 30 responders (11 chronic hepatitis, 1 cirrhosis) who maintained remission up to 1 yr after cessation of interferon treatment. Fourteen patients (7 chronic hepatitis, 7 cirrhosis) with autoantibodies (12 antinuclear, 2 anti-liver-kidney microsomal) were treated with prednisone. The mean viremia level significantly increased after 3 mo of treatment, even in face of ALT decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis C viremia in chronic liver disease: Relationship to interferon-α or corticosteroid treatment

We assessed the pattern of hepatitis C viremia in chronic liver disease by studying 100 hepatitis C virus antibody–positive patients: 48 with chronic hepatitis, 21 with cirrhosis and 31 with hepatocellular carcinoma and cirrhosis. Serum hepatitis C virus RNA was detected by means of both the conventional nested polymerase chain reaction and a newly developed assay based on branched DNA that can also quantify viremia. Hepatitis C virus RNA was found in 94 of 100 patients with polymerase chain reaction and in 71 of 100 patients with branched-DNA (p < 0.001). Mean viremia level (× 103 genome equivalents/ml ± S.D.), as assessed with the branched-DNA test, was 5,700 ± 7,618 in the 48 patients with chronic hepatitis, 3,340 ± 3,633 in the 21 patients with cirrhosis and 1,768 ± 2,770 in the 31 patients with hepatocellular carcinoma (p < 0.02). We also analyzed retrospectively the relationship between viremia and treatment. Fifty-five patients (41 chronic hepatitis, 14 cirrhosis) underwent interferon-α treatment. Mean viremia level was comparable among the 30 responders (5,644 ± 8,207) and the 25 nonresponders (5,519 ± 6,208) to interferon, but it was significantly lower (1,841 ± 1,864) in the 12 of 30 responders (11 chronic hepatitis, 1 cirrhosis) who maintained remission up to 1 yr after cessation of interferon treatment. Fourteen patients (7 chronic hepatitis, 7 cirrhosis) with autoantibodies (12 antinuclear, 2 anti–liver-kidney microsomal) were treated with prednisone. The mean viremia level significantly increased after 3 mo of treatment, even in face of ALT decrease. In conclusion, serum hepatitis C virus RNA is detectable in almost all patients with hepatitis C virus antibody–positive chronic liver disease, polymerase chain reaction being more sensitive than the branched-DNA test. Hepatitis C viremia is lowest in patients with advanced liver disease. A low pretreatment value of viremia could identify patients with chronic hepatitis who will derive long-term benefit from interferon. Corticosteroids generally increase viremia in hepatitis C virus antibody–positive, autoantibody-positive chronic hepatitis, independent of ALT response. (Hepatology 1994;19:273–279).

Quantitation of HCV-replication using one-step competitive reverse transcription-polymerase chain reaction and a solid phase, colorimetric detection method

A solid phase assay for the colorimetric detection of competitively amplified HCV-cDNA has been established and used to investigate clinical samples from patients with chronic hepatitis. The assay is based on the reduction in the amplification of an hepatitis C virus-related competitor molecule by wild-type hepatitis C virus during polymerase chain reaction. The internal standard contains a lac operator sequence, allowing the amount of amplified competitor to be determined using a lac I-repressor/beta-galactosidase fusion protein. The reduction in the amplification of competitor is dependent upon the concentration of HCV-RNA in the original sample. External hepatitis C virus wild-type standards are used to calibrate each concurrently tested set of patients. We present and discuss the potential benefit, but also the limitations of this new approach for quantifying hepatitis C virus viremia. In 47 serum samples from 28 patients with chronic hepatitis C virus infection, including five repeatedly tested alpha HCV positive patients under interferon therapy, viral titer was determined. Sera from nine healthy blood donors served as controls. The sensitivity and specificity of this procedure are identical to those of conventional nested polymerase chain reaction. As both internal and external standards are used in every assay and final detection of amplicons can be carried out in microtiter plates, this reliable and time-saving test system may be routinely applied for monitoring antiviral treatment or for studying the relation of plus- and minus-stranded HCV-RNA in infected tissues.