Rate Of Polymerase Chain Reaction Research Articles

Usefulness of Real-time PCR to DetectMycobacterium tuberculosisand Nontuberculous Mycobacteria

Background: The purpose of this study was to evaluate recently developed real-time polymerase chain reaction (PCR) assay kit to detect Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) in respiratory specimens. Methods: We assessed the positive rate of the real-time PCR assay to detect MTB and NTM in 87 culture-positive specimens (37 sputum, 50 bronchial washing), which were performed real-time PCR by using Real-QTM MTB&NTM Kit from January 2009 to June 2009, at Gyeongsang University Hospital. To compare the efficacy with the TB-PCR assay, we evaluated 63 culture-positive specimens (19 sputum, 44 bronchial washing) for MTB or NTM, which were performed TB-PCR by using ABSOLUTETM MTB II PCR Kit from March 2008 to August 2008. Results: Among 87 specimens tested using real-time PCR, MTB and NTM were cultured in 58 and 29, respectively. The positive rate of real-time PCR assay to detect MTB was 71% (22/31) and 92.6% (25/27) in AFB stain-negative and stain-positive specimens. For NTM, the positive rate of real-time PCR was 11.1% (2/18) and 72.7% (8/11) in AFB stain-negative and stain-positive specimens. Among 63 specimens performed using TB-PCR, MTB and NTM were cultured in 46 and 17, respectively. The positive rate of TB-PCR was 61.7% (21/34) and 100% (12/12) in AFB stain-negative and stain-positive specimens. TB-PCR was negative in all NTM-cultured 17 specimens. Conclusion: TB/NTM real-time PCR assay is useful to differentiate MTB and NTM in AFB stain-positive respiratory specimens and it is as effective in detecting MTB with TB-PCR.

PCR-Mediated Early Diagnosis of Fungal infections in Patients with Living-Donor Liver Transplants

Fungal infections are frequently observed in living-donor liver transplantation (LT) and are correlated with the clinical outcome. Early diagnosis of fungus infection is difficult because of the low positive rate of blood culture and the poor specificity of the (1-3)-� -D-glucan assay for fungi. A polymerase chain reaction (PCR)-based method was developed for the early diagnosis of fungal infection. The PCR-based method consists of a two step PCR amplification from peripheral blood samples; the first PCR which detects all species of fungi excluding Mucor, Fusarium and Sporothrix (fungi-specific PCR). Subsequently, nested PCRs is used to determine the species of fungi (species-specific PCR), Aspergillus/Penicillium (ASP/PEN), Candida albicans/parapsilosis/tropicals/ guliermondi (CAN), Candida glabrata (C.glab.), and broad spectrum of fungi (Broad spectrum). This method was applied to peripheral blood samples from 31 LT patients, 11 samples from febrile patients in hematological ward (HW), and 15 patients with fever of unknown origin in the general ward (GW). Fourteen of the LT patients were tested positive with the fungi-specific PCR, 22 with the species-specific PCR. The positive rates of species-specific PCR were 12.9% for ASP/PEN, 29.0% for CAN, 9.7% for C.galb. and 45.2% for the broad spectrum, respectively. Five of the HW patients tested positive with the fungus specific- PCR, and 6 with the species-specific PCR. Two of the GW patients were positive with the fungi-specific-PCR. The fungus-specific PCR has higher specificity than the (1-3)-bete-D-glucan measurement, and the clinical outcome of LT patients correlated significantly with the results of this method. In conclusion, that the newly developed PCR-based method might be useful for early detection and diagnosis of fungus infection in LT patients.

Screening Pneumocystis carinii pneumonia in non–HIV-infected immunocompromised patients using polymerase chain reaction

We evaluated the clinical value of polymerase chain reaction (PCR) for screening Pneumocystis carinii pneumonia (PCP) in non–HIV-infected immunocompromised patients. We retrospectively analyzed PCR and Grocott's methenamine silver (GMS) staining on 1044 clinical specimens obtained from 756 patients. Positive rates of PCR and GMS staining in sputum specimens were 21.1% and 9.5%, respectively ( P < 0.01), and in bronchoalveolar lavage (BAL), specimens were 31.9% and 25.5%, respectively. Among 5 patient groups, the highest GMS staining and PCR positive rates were observed in immunosuppressed patients. In 28 GMS staining-positive patients, positive rates of PCR and GMS staining after a short anti-PCP treatment were 39.3% and 17.9%, respectively. In 5 patients with positive PCR but negative GMS staining results in initial sputum examination, GMS staining result turned positive in subsequent BAL specimens. In conclusion, PCR is sensitive for detection of Pneumocystis in sputum specimens and is useful for screening PCP in non–HIV-infected high-risk patients.

Noninvasive recovery and detection of possum Trichosurus vulpecula DNA from bitten bait interference devices (WaxTags)

The brushtail possum is a major agricultural and ecological pest in New Zealand. A novel noninvasive DNA sampling tool for detecting its presence (WaxTags, or WT) was tested. DNA was recovered from saliva left on WT, and two lengths (407 bp and 648bp) of the cytochrome c oxidase I (COI) barcoding region were amplified by polymerase chain reaction (PCR). PCR products were considered (+) when a DNA band was clearly visible by electrophoresis. Different factors that might affect PCR (+) were investigated with captive possums: (i) both extraction protocols of the QIAGEN DNeasy Blood and Tissue Kit, (ii) effect of an overnight or longer delay of up to 3 weeks before DNA extraction on both COI amplicons, and (iii) effect of the individual, order and magnitude of the bite. Extraction protocols were not significantly different. The effect of the overnight delay was not significant, and amplification of the short amplicon was significantly higher (100%) than for the long fragment (48%). After a two or 3-week delay, the short amplicon had 94% and 56% PCR (+), success rates, respectively. Individual, order and magnitude of a bite had no significant effect. The delay trial was repeated with WT from the wild, for which PCR (+) rate of the short amplicon was 63%, regardless of freshness. Four microsatellites were amplified from captive WT samples. We conclude that DNA from saliva traces can be recovered from WT, a potential new tool for noninvasive monitoring of possums and other wildlife.

Predicting failure rate of PCR in large genomes

We have developed statistical models for estimating the failure rate of polymerase chain reaction (PCR) primers using 236 primer sequence-related factors. The model involved 1314 primer pairs and is based on more than 80 000 PCR experiments. We found that the most important factor in determining PCR failure is the number of predicted primer-binding sites in the genomic DNA. We also compared different ways of defining primer-binding sites (fixed length word versus thermodynamic model; exact match versus matches including 1–2 mismatches). We found that the most efficient prediction of PCR failure rates can be achieved using a combination of four factors (number of primer-binding sites counted in different ways plus GC% of the primer) combined into single statistical model GM1. According to our estimations from experimental data, the GM1 model can reduce the average failure rate of PCR primers nearly 3-fold (from 17% to 6%). The GM1 model can easily be implemented in software to premask genome sequences for potentially failing PCR primers, thus improving large-scale PCR-primer design.

Low Resistance to Adefovir Combined With Lamivudine: A 3-Year Study of 145 Lamivudine-Resistant Hepatitis B Patients

Adefovir monotherapy is an established treatment modality for lamivudine-experienced patients with chronic hepatitis B, but it carries a significant risk of resistance in the long term. We assessed whether this risk could be overcome by adefovir-lamivudine combination therapy. A total of 145 lamivudine-resistant patients with chronic hepatitis B (73% cirrhotics, 86% hepatitis B e antigen negative, 92% genotype D) were treated with adefovir 10 mg in addition to lamivudine 100 mg. Liver function tests and hepatitis B virus (HBV) DNA (Versant 3.0) were assessed bimonthly, whereas adefovir-related mutations were searched by INNO-LiPA assay at baseline and at yearly intervals. During 42 months (range, 12-74), 116 patients (80%) cleared serum HBV DNA, 67 (84%) had normalized alanine aminotransferase levels, and 145 (100%) remained free of virologic and clinical breakthroughs, independently of the degree of HBV suppression. The rtA181V/T was the only adefovir-related mutation detected, which occurred in 6 patients at baseline (4%; 1 rtA181V and 5 rtA181T) and in an additional 3 patients (2%; all rtA181T) during treatment. In all these 9 patients, HBV DNA levels progressively declined during therapy to become undetectable in 7 (78%). The 1-, 2-, 3-, and 4-year cumulative rates of de novo rtA181T were 1%, 2%, 4%, and 4%, respectively. None of the cirrhotic patients clinically decompensated, but 11 (12%) developed hepatocellular carcinoma. Under prolonged adefovir-lamivudine therapy, patients with lamivudine-resistant hepatitis B were unlikely to develop genotypic resistance to adefovir and had durable prevention of virologic and clinical breakthrough.

Detection of Staphylococcus aureus in Dairy Products by Polymerase Chain Reaction Assay

Abstract A polymerase chain reaction (PCR) assay was employed for direct detection of Staphylococcus aureus without enrichment in dairy products. A solvent extraction procedure was successfully modified for the extraction of Staphylococcus aureus DNA from artificially contaminated whole milk, skim milk, and cheese. A primer targeting the thermostable nuclease gene ( nuc) was used in the PCR. A DNA fragment of 279 bp was amplified. The PCR product was confirmed by DNA sequencing. In this study, the PCR, GB-4789.10–94, Perifilm RSA. Count Plate, and Baird-Parker + RPF Agar were compared. The sensitivity of the PCR was 10 CFU mL −1 of whole milk, skim milk, and 55 CFU g −1 of cheese. The developed methodology allowed for detection of Staphylococcus aureus in dairy products in less than 6 h. The time taken for the development of this PCR assay was 12–24 h, less than the time taken by the general PCR assay using the enrichment method, and the coincidence rate of this developed PCR was 94.3%, the sensitivity was 100%. It was a rapid, sensitive, and effective method for PCR to detect Staphylococcus aureus in milk and milk products.

Real-time TaqMan polymerase chain reaction assays for quantitative detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum

Evidence has been presented that the species currently known as Ureaplasma urealyticum should be separated into 2 species— Ureaplasma parvum (previously, U. urealyticum biovar 1) and U. urealyticum (previously, U. urealyticum biovar 2). Differentiation and quantification of U. parvum and U. urealyticum can provide important information of the epidemiology of Ureaplasma infections. We developed 2 real-time TaqMan polymerase chain reaction (PCR) assays that would allow rapid, specific, sensitive, quantitative detection and convenient differentiation of U. parvum and U. urealyticum. One hundred twenty-eight clinical specimens were studied and compared with results obtained by culture methods and conventional PCR. The positive rate of real-time TaqMan PCR (59.4%, 76 of 128) was higher than that of culture methods (42.2%, 54 of 128) and conventional PCR (50%, 64 of 128). Of 76 positive specimens, 86.8% (66) contained U. parvum only, 10.5% (8) contained U. urealyticum only, and 2.6% (2) contained both. The copy numbers of 11 positive specimens were in the range of 10 1 to 10 3 copies per reaction mixture, 18 in the range of 10 3 to 10 5, and 47 in the range of 10 5 to 10 8. In the future, quantitative detection and convenient differentiation of real-time TaqMan PCR assays will assist in the study of the pathogenesis and epidemiology of Ureaplasma infections.

Detection of Viral Deoxyribonucleic Acid in Amniotic Fluid: Association with Fetal Malformation and Pregnancy Abnormalities

Objective: To first test the hypothesis that the presence of viral nucleic acid in amniotic fluid (AF) is associated with an abnormal pregnancy outcome, and second, to determine if the overall rate of polymerase chain reaction (PCR) positivity and the distribution of virus types vary geographically. Study Design: Cytomegalovirus (CMV), parvovirus B19, adenovirus, enterovirus, herpes simplex virus, Epstein-Barr virus, and respiratory syncytial virus nucleic acids were sought in 423 AF samples obtained for clinical indications: 284 from the East Coast (EC) and 139 from the Midwest (MW). Results: Gestational age at sampling was 19.1 weeks for EC and 20.1 weeks for MW. 13.5% of karyotypically normal singleton pregnancies (57/423) had a positive AF PCR. 11% of AF PCR from the EC while 18% of AF PCR from the MW were positive (p = 0.06). The most commonly detected viruses were adenovirus (77%), enterovirus (12%), and CMV and parvovirus B19 (5% each). Twenty-four percent of sonographically abnormal pregnancies (33/136) had a positive AF PCR compared to only 8.4% (24/287) of normal pregnancies (p < 0.001). Conclusion: A positive AF PCR is associated with an increased rate of fetal structural malformations, intrauterine growth restriction, hydrops and other fetal abnormalities. There were no significant geographic differences in the incidence of AF viral PCR positivity.

Enrichment polymerase chain reaction for the detection of Ki-ras mutations: relevance of Taq polymerase error rate, initial DNA copy number, and reaction conditions on the emergence of false-positive mutant bands.

Screening for oncogene mutations as a marker for malignancy can be a powerful tool for the early diagnosis of cancer. The enrichment polymerase chain reaction (PCR) is a sensitive method for the detection of low-frequency mutations in small samples. However, false-positive results, caused by methodological errors, may have severe clinical implications. When applied to the detection of Ki-ras mutations in pancreatic secretions, the assay sensitivity is limited to approximately 1:1400. Our investigation of Ki-ras mutations in blood samples from patients with pancreatic carcinoma revealed PCR bands presumably derived from mutant Ki-ras in samples from healthy volunteers, while all blood samples of the patients with pancreatic carcinomas showed a wild-type band pattern. Mathematical modeling of the PCR reaction reveals that the rate of false positive PCR results depends on the initial amount of DNA, the Taq polymerase error rate, the number of PCR reaction cycles, reaction efficiency and the restriction endonuclease chosen. The overall error rate of false positive results of the enrichment PCR can be reduced to the square of the rate of a single-step analysis if repeated amplifications of the same DNA specimen show an identical result.

Use of a PCR assay for the specific and sensitive detection of Trypanosoma Spp. in naturally infected dairy cattle in peri-urban Kampala, Uganda.

The objective of this study was to compare the sensitivity and specificity of the polymerase chain reaction (PCR) with the hematocrit centrifugation technique (HCT) and the mini-anion-exchange centrifugation technique (m-AECT) for diagnosis of trypanosome infections in livestock. In a cross-sectional study, 486 cattle from 50 randomly selected farms in Mukono County, Uganda were investigated in June 1994. The direct parasitological techniques were performed in the field, resulting in 45 (9.3%) animals positive by HCT and 78 (16%) positive by m-AECT. The total prevalence (combined evidence of HCT and m-AECT) was 18.9%, with 78.2% Trypanosoma brucei only, 10.9% T. vivax and 10.9% mixed (T. bruceil T. vivax) infections. Trypanosomes of the subgenus Nannomonas were not detected. DNA was prepared by lysis from 181 randomly selected blood samples and amplified by PCR using species-specific oligonucleotide primers. Overall, the PCR gave positive results in 63 (34.8%) blood samples, with 76.2% positive only for T. brucei, 20.6% positive only for T. vivax and 3.2% positive for mixed (T. bruceil T. vivax) infections. The preliminary results from this study demonstrate that the detection rate of PCR is about two times higher than that of the direct parasitological techniques, suggesting a higher sensitivity. The higher proportion of T. vivax infections detected by PCR as compared to HCT/m-AECT is likely to be due to false parasitological classifications which might occur under field conditions.

Monitoring of Pulmonary Tuberculosis by Polymerase Chain Reaction After Antituberculous Treatment

BACKGROUND: As living and dead Mycobacteria could be amplified by polymerase chain reaction(PCR), it was considered that PCR was inappropriate for the monitoring of pulmonary tuberculosis after treatment. But we found negative conversion of PCR after successful treatment. We would like to know about the negative conversion rate of PCR and its conversion time after antituberculous treatment. METHODS: We collected 113 sputums from the 16 patients of pulmonary tuberculsosis visiting Catholic University Hospital of Taegu Hyosung. We consecutively tested AFB smear, AFB culture and PCR by 2 to 4 weeks after antituberculous therapy. The patients were classified according 13 the chest X ray findings. RESULTS: We detectsd negative conversion of PCR from all 16 patients of the pulmonary tuberculosis within 30 weeks after treatment. The average negative conversion time was 16 +/- 8 weeks. The conversion time according to the chest X-ray findings were as follows : For the 8 cases of minimum were 9 +/- 5 weeks,4 cases of modreate advanced were 20 +/- 8 weeks, and 4 cases of far advanced were 23 +/- 2 weeks. The product of PCR was gradually decreased according to the duration of treatment. CONCLUSIONS: From the results of our study, we could utilize M. tubercuosis PCR for the prediction of therapy response and monitoring of the patient with pulmonary tuberculosis after treatment.

Detection of Ureaplasma urealyticum in urine of patients with systemic lupus erythematosus and healthy individuals by culture and polymerase chain reaction.

A method was developed to detect Ureaplasma urealyticum in urine by the polymerase chain reaction (PCR). A 457-bp fragment of the urease gene of U. urealyticum was amplified by PCR. Before PCR, components disturbing the amplification had to be reduced. This was possible by diluting the urine 1 in 10 with distilled water and by the extraction of the U. urealyticum DNA. Urine specimens from 41 patients with systemic lupus erythematosus (SLE) and 21 healthy individuals were treated by the dilution method and investigated by PCR for U. urealyticum DNA. The results were compared with those obtained by culture and the detection rates of PCR and culture were found to be identical. Also there was no difference in the detection rates of U. urealyticum from urine of SLE patients and healthy individuals; 10 (24.4%) of the 41 urine specimens from SLE patients and five (23.8%) of the 21 urine specimens from healthy individuals gave positive results for U. urealyticum. The results of this study do not indicate a decisive role for U. urealyticum in SLE.

Measurement by the polymerase chain reaction of the Epstein‐Barr virus load in infectious mononucleosis and AIDS‐related non‐Hodgkin's lymphomas

A polymerase chain reaction (PCR) assay for the detection of Epstein-Barr virus (EBV) sequences in various clinical samples, especially peripheral blood leukocytes (PBL) and serum, was carried out and the results obtained were compared with specific EBV serology. One hundred seventy patients were enrolled in the study: 89 healthy blood donors, 22 asymptomatic patients, 36 individuals with primary EBV infection (including 19 patients with infectious mononucleosis [IM]), 22 HIV-infected subjects (including 4 with hairy oral leukoplakia, 3 with central nervous disorders, and 15 with non-Hodgkin's lymphoma). All the serum samples from the healthy blood donors were negative. In patients with IM and in AIDS-non Hodgkin's lymphoma (ARNHL), PCR was strongly positive in leukocytes (> 2,000 genome equivalents/10(4) cells), which was correlated with detectable amounts of EBV DNA in serum. The overall positivity rate of PCR in serum was 58.8%, 68%, and 73% of cases for non-IM primary EBV infections, IM, and ARNHL, respectively. In two cases of EBV primary infection, the viral DNA was detected in serum, respectively 1 month and 2 months before IgM positivity and IgG rise. In one case of ARNHL followed up for several months, PCR (viral load of 2,000 genome equivalents/10(4) cells) became positive concurrently with appearance of lymphoma. In immunocompromised individuals, PCR EBV, if carried out in larger prospective studies, could be considered as a tumor marker, useful for predicting EBV-driven lymphoma and follow-up therapy.

Identification of Mycobacterium tuberculosis in Pleural Effusion by Polymerase Chain Reaction(PCR)

Background: Since polymerase chain reaction(PCR) was devised by Saiki in 1985, it has been used extensively in various fields of molecular biology. Clinically, PCR is especially useful in situation when microbiological or serological diagnosis is limited by scanty amount of causative agents. Thus, PCR can provide rapid and sensitive way of detecting M. tuberculosis in tuberculosis pleurisy which is diagnosed in only about 60 % of cases by conventional method. Method: To evaluate the diagnostic usefulness of PCR in tuberculosis pleurisy, The results of PCR was compared with those of conventional method, including pleural biopsy. The pleural effusion fluid was collected from 7 proven patients, 7 clinically suspected patients and control group(7 patients with malignant effusion). We extracted DNA from pleural fluid by modified method of Eisennach method(1991). The amplification target for PCR was 123 base pair DNA, a part of IS6110. Result: 1) Sensitivity of PCR: We detected upto 50fg DNA. 2) In patients with pleural effusion of proven tuberculosis, the positive rate of PCR was 85.7%(6/7). In patients with pleural effusion of clinically suspected tuberculosis, the positive rate was 71.5%(5/7). In control group, positive rate was 0%(0/7). Conclusion: We concluded that PCR method could be a very rapid, sensitive and specific one for diagnosis of M tuberculosis in pleural effusion. Further studies should be followed for the development of easier method.

Detection of mycobacterial DNA in sarcoidosis and tuberculosis with polymerase chain reaction

The cause of sarcoidosis is unknown. However, the histological similarity between the disorder and tuberculosis suggests that mycobacteria might contribute to the pathogenesis of sarcoidosis. We have used the polymerase chain reaction (PCR) to detect mycobacterial DNA in clinical samples from patients with sarcoidosis. 104 patients were included in the study (62 referred for possible tuberculosis and 20 for possible sarcoidosis, and 22 control patients who had undergone bronchoscopy for other reasons). Bronchoalveolar lavage samples, bronchial washings, and tissue specimens (1 from each patient) underwent assay by PCR as well as bacteriological, histological, and cytological examination. We used two PCR reactions: in the first the complex-specific insertion sequence IS986/ IS6110 was used to specifically detect DNA from Mycobacterium tuberculosis complex bacteria; in the second, conserved sequences of the mycobacterial groEL gene were used to detect DNA from mycobacteria other than M tuberculosis. The PCR was more sensitive than culture for diagnosis of tuberculosis. However, the false-positive PCR rate for M tuberculosis was 9%. M tuberculosis DNA was found in half the sarcoidosis patients, and non-tuberculosis mycobacterial DNA in a further 20%. The findings that a significant proportion of the sarcoidosis patients in this study have mycobacteria in their lungs and that most of these mycobacteria belong to M tuberculosis complex suggest an aetiological role for mycobacteria in sarcoidosis.