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PubMed2001

Cross-protection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and RT-PCR and nested PCR for their detection.

A 1-step RT-PCR assay, targeting a 730 bp fragment of the nucleocapsid (N) gene of bovine coronavirus (BCV), and a nested PCR assay, targeting a 407 bp fragment of the N gene, were developed to detect BCV in nasal swab and fecal samples of calves experimentally exposed to BCV. Both 1-step RT-PCR and nested PCR recognized cell culture passaged isolates of 10 bovine respiratory coronavirus (BRCV), 5 calf diarrhea (CD) and 8 winter dysentery (WD) strains of BCV, but not transmissible gastroenteritis coronavirus or bovine rotavirus. The sensitivity of the 1-step RT-PCR and nested PCR was compared to that of an antigen-capture ELISA. The lowest detection limit of the 1-step RT-PCR and nested PCR as determined by using tenfold serial dilutions of the BRCV 255 and 440 strains in BCV negative nasal swab suspensions from preexposure gnotobiotic calves was 2 x 10(4) and 2 x 10(2) TCID50/0.1 ml for each strain, respectively. The lowest detection limit of the antigen-capture ELISA as determined by using the same serially diluted samples was 1 x 10(6) TCID50/0.1 ml for each strain. Therefore, the 1-step RT-PCR and nested PCR assays were 50 and 5000 times, respectively more sensitive than the antigen-capture ELISA to detect BRCV in nasal swab suspensions. To investigate in vivo cross-protection between the BRCV and CD or WD strains of BCV and to detect nasal and fecal shedding of BCV using the 1-step RT-PCR, nested PCR and antigen-capture ELISA, 6 colostrum-deprived and two gnotobiotic calves were inoculated with a BRCV, a CD or a WD strain of BCV and then challenged 3-4 weeks later with either BRCV, CD or WD strains of BCV. All calves developed diarrhea after inoculation and BCV antigen (ELISA) or RNA (RT-PCR) was detected in the diarrheic fecal samples or the corresponding nasal swab samples. In addition, low amounts of BCV were also detected only by nested PCR in the fecal and nasal swab samples before and after diarrhea. No respiratory clinical signs were observed during the entire experimental period, but elevated rectal temperatures were detected during diarrhea in the BCV-inoculated calves. All calves recovered from infection with the BRCV, CD, or WD strains of BCV were protected from BCV-associated diarrhea after challenge exposure with either a heterologous or homologous strain of BCV. However, all calves challenged with heterologous BCV strains showed subclinical BCV infection evident by detection of nasal and fecal shedding of BCV RNA detected only by nested PCR. Such results confirm field and experimental data documenting reinfection of the respiratory and enteric tracts of cattle, suggesting that, in closed herds, respiratory or enteric tract reinfections may constitute a source of BCV transmissible to cows (WD) or neonatal or feedlot calves. In addition, the present 1-step RT-PCR and nested PCR assays were highly sensitive to detect BCV in nasal swab and fecal specimens. Therefore, these assays should be useful to diagnose BCV infections in calves and adult cows.

Animals↗

PubMed2004

Comparison of PCR-ELISA and LightCycler real-time PCR assays for detecting Salmonella spp. in milk and meat samples.

In a previous study, we reported the performance of a PCR assay amplifying 285-bp of the invA gene of Salmonella spp. through an international ring-trial involving four participating laboratories [Int. J. Food Microbiol. 89 (2003) 241]. Based on the validated set of primers and recent advancements in PCR technology, we have designed two specific PCR assays for detecting Salmonella spp. We have compared PCR-enzyme-linked immunosorbent assay (PCR-ELISA) and LightCycler real-time PCR assay (LC-PCR) with the standard ISO 6579 bacteriological reference method. The two PCR tests incorporated an internal amplification control (IAC) co-amplified with the invA gene of Salmonella to monitor potential PCR inhibitors and ensure successful amplification. The selectivity study involved 84 Salmonella and 44 non-Salmonella strains and the samples tested were represented by 60 artificially-contaminated samples of fish, minced beef and raw milk, and 92 naturally-contaminated milk and meat samples. When using either PCR-ELISA or LC-PCR assays, only Salmonella strains were detected. PCR-ELISA and LC-PCR assays gave with pure Salmonella cultures the same detection limit level of 10(3)CFU/ml, which corresponds respectively to 50 and 10 cells per PCR tube. Data on artificially contaminated samples indicated that both PCR methods were able to detect after enrichment less than five Salmonella cells in 25 g of food, giving 100% concordance with the ISO 6579 reference method. The results on naturally contaminated samples demonstrated that despite certain inhibition problems, LC-PCR and PCR-ELISA assays were highly specific and sensitive, and provide a powerful tool for detection of Salmonella in food samples.

Animals↗

PubMed2003

Evaluation of the use of PCR and reverse transcriptase PCR for detection of pathogenic bacteria in biosolids from anaerobic digestors and aerobic composters.

A PCR-based method and a reverse transcriptase PCR (RT-PCR)-based method were developed for the detection of pathogenic bacteria in organic waste, using Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and Staphylococcus aureus as model organisms. In seeded organic waste samples, detection limits of less than 10 cells per g of organic waste were achieved after one-step enrichment of bacteria, isolation, and purification of DNA or RNA before PCR or RT-PCR amplification. To test the reproducibility and reliability of the newly developed methods, 46 unseeded samples were collected from diverse aerobic (composting) facilities and anaerobic digestors and analyzed by both culture-based classical and newly developed PCR-based procedures. No false-positive but some false-negative results were generated by the PCR- or RT-PCR-based methods after one-step enrichment when compared to the classical detection methods. The results indicated that the level of activity of the tested bacteria in unseeded samples was very low compared to that of freshly inoculated cells, preventing samples from reaching the cell density required for PCR-based detection after one-step enrichment. However, for Salmonella spp., a distinct PCR product could be obtained for all 22 nonamended samples that tested positive for Salmonella spp. by the classical detection procedure when a selective two-step enrichment (20 h in peptone water at 37 degrees C and 24 h in Rappaport Vassiliadis medium at 43 degrees C) was performed prior to nucleic acid extraction and PCR. Hence, the classical procedure was shortened, since cell plating and further differentiation of isolated colonies can be omitted, substituted for by highly sensitive and reliable detection based on nucleic acid extraction and PCR. Similarly, 2 of the 22 samples in which Salmonella spp. were detected also tested positive for Listeria monocytogenes according to a two-step enrichment procedure followed by PCR, compared to 3 samples that tested positive when classical isolation procedures were followed. The study shows that selective two-step enrichment is useful when very low numbers of bacterial pathogens must be detected in organic waste materials, such as biosolids. There were no false-positive results derived from DNA of dead cells in the waste sample, suggesting that it is not necessary to perform RT-PCR analyses when PCR is combined with selective enrichment. Large numbers of added nontarget bacteria did not affect detection of Salmonella spp., L. monocytogenes, and Y. enterocolitica but increased the detection limit of Staphylococcus aureus from <10 to 10(4) CFU/g of organic waste. Overall, the detection methods developed using seeded organic waste samples from one waste treatment facility (WTF) needed to be modified for satisfactory detection of pathogens in samples from other WTFs, emphasizing the need for extensive field testing of laboratory-derived PCR protocols. A survey of 13 WTFs in Germany revealed that all facilities complied with the German Biowaste Ordinance, which mandates that the end product after anaerobic digestion or aerobic composting be free of Salmonella In addition, all biosolids were free of L. monocytogenes, Staphylococcus aureus, and Y. enterocolitica, as evidenced by both classical and PCR-based detection methods.

Bacteria↗

PubMedDate not supplied

"Single step" PCR with a sensitivity similar to nested PCR for the detection of hepatitis C virus RNA.

OBJECTIVE: Evaluation of the performance of different HCV PCR detection systems for HCV RNA: A nested PCR, considered the reference assay, was compared with two single-step methods (ss-PCR): the first is based on the detection of PCR products by liquid hybridization with a 32P end-labelled probe (isotopic ss-PCR), while the second assay is a colorimetric method (colorimetric ss-PCR) using microwell plate hybridization with a specific nucleic acid probe (Amplicor HCV PCR, Roche Diagnostics Systems). METHODS: Sera from 56 patients with suspected hepatitis C infection based on reactive serology or altered liver parameters, and sera from 15 blood donors were tested for HCV RNA: After RNA extraction, the synthesized HCV cDNA was amplified in parallel using isotopic ss-PCR, colorimetric ss-PCR and nested PCR. The products were detected by autoradiography, color development and ethidium bromide fluorescence, respectively. RESULTS: In order to assess the analytical sensitivity of ss-PCR versus that of nested of PCR, experiments included serial dilutions of positive control samples. Results showed that both methods had an extinction signal at the 1:512 dilution. A comparative analysis of 71 clinical sera samples was obtained using the three protocols and the results clearly documented 100% concordance. CONCLUSIONS: Single step PCR methods for HCV RNA have a sensitivity equal to that of nested PCR and appear more suitable for diagnostic applications. Ss-PCR is safer than nested PCR in terms of both specificity and contamination problems. In particular, the Roche Amplicor HCV PCR assay minimizes sample exposure and management problems.

Hepacivirus↗

PubMed2005

Detection of Mycoplasma genitalium in urogenital specimens by real-time PCR and by conventional PCR assay.

A real-time LightCycler PCR (LC-PCR) with hybridization probes for detection of Mycoplasma genitalium in endocervical and first void urine specimens was developed and compared to a conventional PCR. The primers for both assays were identical and designed to amplify a 427 bp fragment of the 16S rRNA gene of M. genitalium. The LC-PCR assay had a detection limit of < 5 bacterial genomes per reaction when dilutions of genomic DNA from a type strain of M. genitalium were tested. First void urine from 398 men and first void urine and endocervical specimens from 301 women attending an STD clinic were analysed by LC-PCR and by the conventional PCR. Using the conventional PCR as reference, the LC-PCR had a specificity of 99.7 % and a sensitivity of 72.2 % for the detection of M. genitalium in first void urine samples from men. There was no significant difference in the performance of the LC-PCR assay compared to the conventional PCR when endocervical swabs were considered (58 and 65 %, respectively) or with a set of endocervical swab/urine specimens for which the LC-PCR assay detected 73 % of the infections (specificity = 98.6 % and sensitivity = 68.2 %) while the conventional PCR detected 85 % of the infections. With female urine specimens there was a significant difference between the two assays (38 and 73 %, respectively; P = 0.01 McNemar's test). This illustrates the need to analyse both endocervical and urine specimens, because M. genitalium DNA was detected in only one of the two specimens in a great number of the M. genitalium-infected women. The lower sensitivity of the LC-PCR assay was probably caused by a combination of inhibition and limitations regarding the amount of template DNA. The LC-PCR assay was easy to perform and the simultaneous amplification and detection eliminated the need for further handling of PCR products. With improvement in sample preparation methods and increased volumes of the template DNA, the LC-PCR assay could be a useful routine diagnostic method.

Adolescent↗

PubMed2002

Nuclear DNA PCR-RFLPs that distinguish African and European honey bee groups of subspecies. I: Comparison of long PCR and standard PCR to screen for polymorphisms.

Nuclear DNA RFLPs betweenAfrican and European honeybees (Apis mellifera L.) were sought by amplifying short (1-2 kbp) and long (> 5 kbp) anonymous regions of DNA and digesting the respective PCR products with a collection of restriction enzymes. Three short and three long regions were each screened with 26-31 enzymes. From a total of 163 locus enzyme combinations (LECs), seven revealed informative polymorphisms. One of these LECs came from one of the three short regions (S-3 with AluI), producing a total of seven alleles, five of which were African-specific. The search for useful RFLPs was far more effective within the long regions. The other six inforrmative LECs came from the three long regions (L-1 with AluI, L-2 with AvaI and HaeIII, and L-5 with HaeIII, DdeI, and SpeI), producing a total of 43 alleles, of which 18 were African-specific, 13 were European-specific, and two were predominantly found in the European samples. Among the European alleles, two were predominantly found in west European honey bee subspecies. Strong associations between alleles generated by pairs of enzymes at a locus were found.

Africa↗

PubMed1996

Evaluation of PCR and nested PCR for laboratory diagnosis of hepatitis C virus infection.

The detection of hepatitis C virus (HCV) RNA by nested polymerase chain reaction (PCR) is believed to be the most reliable method to diagnose HCV infections. A pitfall of nested PCR is that it is prone to contamination. Single step reverse transcription-PCR (RT-PCR) was performed, prospectively, on 80 sera from 59 patients with a set of primers that amplified a 273 bp sequence unique to the 5' noncoding (NC) region of the HCV genome. Nested PCR, was performed on all PCR negative specimens with a set of primers that amplified a 255 bp internal to the original primers. Single step RT-PCR was positive on 45 sera from 35 patients following gel electrophoresis and on two additional sera from two patients following Southern blot hybridization. Nested PCR was positive on two more sera following gel electrophoresis of the nested PCR products. These two patients were seropositive and subsequent serum from one patient was positive by single step PCR. Three additional sera were positive following Southern blot analysis of the nested PCR products. Two patients were seropositive and had elevated serum alanine aminotransferase (ALT) levels. The third patient was seronegative with normal ALT level and was considered a false positive. The remaining seronegative control specimens were PCR negative by both methods. The majority of PCR positive patients (82%) had elevated ALT levels, while the majority of PCR negative seropositive patients had normal ALT levels. We conclude that single step PCR is a sensitive test for the laboratory diagnoses of the majority of the HCV infections.

Animals↗

PubMed2006

Real-time PCR for mumps diagnosis on clinical specimens--comparison with results of conventional methods of virus detection and nested PCR.

BACKGROUND: Since November 2003, the UK has seen a dramatic rise in the number of mumps cases, resulting in increasing demands on virology laboratories to confirm mumps infection in a timely and efficient manner. Traditional mumps virus detection methods are often insensitive, lengthy, and cumbersome. Some laboratories in the UK now use molecular methods that are based on nested polymerase chain reaction (PCR). Early serological diagnosis often relies on detection of anti-mumps IgM, which may be absent in the first 10 days of illness. OBJECTIVES: We compared a one-step real-time RT-PCR with an established nested PCR (SH-PCR) and virus detection by culture and antigen detection, and assessed the clinical usefulness of mumps real-time PCR for diagnosis from CSF. STUDY DESIGN: In total, 280 clinical samples were investigated by real-time PCR, nested PCR and a combination of traditional virus detection methods (antigen detection on oral samples, cell culture on all samples). Furthermore, 88 CSF samples submitted for diagnosis of possible viral meningitis were analysed by real-time PCR. RESULTS: The real-time PCR detected the highest number of positive oral samples (119/180) compared to SH-PCR (92/180) and combined virus culture and antigen detection procedures (90/180). Sensitivity of mumps virus detection in urine was poor for all three methods: 34.0% (traditional detection), 29.8% (real-time PCR) and 2.1% (SH-PCR), respectively. Real-time PCR on 88 CSF samples identified five patients with mumps meningitis, significantly increasing viral diagnosis in this cohort. CONCLUSION: Real-time PCR on oral samples is the investigation of choice for mumps infection. Mumps virus detection in urine by any of the PCRs used was clearly less successful. Real-time PCR on CSF samples seems a promising adjunct for diagnosis of mumps meningitis, especially in an age group with high incidence of mumps.

Cohort Studies↗

PubMed2003

Comparison of AP-PCR typing and PCR-ribotyping for estimation of nosocomial transmission of Clostridium difficile.

We recently attempted to clarify an increased incidence of Clostridium difficile-associated diarrhoea (CDAD) in our hospital by arbitrarily primed polymerase chain reaction (AP-PCR) typing of isolates from 147 consecutive patients collected during a 12 month period (Wullt et al. J Hosp Infect 1999;43:265-273). In the present study we compared the results based on previous AP-PCR data with those based on recent PCR ribotyping of the same isolates and re-analysis of a subset of isolates by AP-PCR typing. The pattern of PCR ribotypes was similar among inpatients and outpatients. A cluster of three closely related PCR ribotypes, related to those of the serogroup H and A8 type strains, dominated and comprised 31% of inpatient and 28% of outpatient C. difficile isolates. The apparent nosocomial transmission rate among inpatients with CDAD was only 9% by AP-PCR typing compared with 18 or 36% by PCR ribotyping depending on the definition used (proportion of patients sharing C. difficile type and ward within two or 12 months). Corresponding rates for all CDAD patients were 5% by AP-PCR and 11 or 21% by PCR ribotyping. Thus, most CDAD patients apparently became ill due to their endogenous strain of C. difficile. Because of the low concordance between the two typing methods the proportion of patients fulfilling the criteria for nosocomial transmission by both methods was only 1%. Re-examination of isolates from patients with recurrences revealed a reproducibility problem with AP-PCR typing. We conclude, that of these two PCR-based options for typing of C. difficile PCR ribotyping offers a superior experimental robustness compared with AP-PCR typing.

Clostridioides difficile↗

PubMed2004

Comparison between LightCycler Real-Time Polymerase Chain Reaction (PCR) assay with serum and PCR-enzyme-linked immunosorbent assay with whole blood samples for the diagnosis of human brucellosis.

BACKGROUND: To overcome some of the limitations of conventional microbiological techniques, polymerase chain reaction (PCR)-based assays have been proposed as a useful tool for the diagnosis of human brucellosis. METHODS: A single-blinded comparative study was undertaken that compared 2 different PCR assays: a SYBR Green I LightCycler-based Real-Time PCR assay (LC-PCR; Roche Diagnostic) with serum samples and a PCR-enzyme-linked immunosorbent assay (ELISA) with whole blood samples. Both assays amplify a 223-bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for Brucella genus (BCSP31). We analyzed the diagnostic yield of these assays with 60 samples obtained from patients with active brucellosis and 37 samples obtained from a control group composed of patients with febrile syndromes of other defined etiologies, asymptomatic subjects with past brucellosis or exposure to Brucella infection who had persistently high titers of anti-Brucella antibodies, and healthy subjects. RESULTS: The sensitivities of LC-PCR with serum samples, PCR-ELISA with whole blood samples, and blood cultures were 93.3%, 90%, and 65%, respectively. Three control samples (8.1%) had a positive PCR-ELISA result, and 2 of these samples (5.4%) also had positive LC-PCR results. The specificity and positive likelihood ratios were 94.6% and 17.3, respectively, for LC-PCR and 91.9% and 11.1, respectively, for PCR-ELISA. CONCLUSIONS: The diagnostic yield of LC-PCR with serum samples was higher than that of PCR-ELISA with whole blood samples. The speed and technical simplicity of LC-PCR in serum samples make it a useful alternative to blood cultures for patients with suspected brucellosis and negative or doubtful serological test results.

Adolescent↗

PubMed2000

Comparison of PCR-ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for typing Clostridium difficile.

Clostridium difficile is now recognized as the major agent responsible for nosocomial diarrhea in adults. Among the genotyping methods available, arbitrarily primed PCR (AP-PCR), PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) have been widely used for investigating outbreaks of C. difficile infections. However, the comparative typing ability, reproducibility, discriminatory power, and efficiency of these methods have not been fully investigated. We compared the results of three methods-AP-PCR with three different primers (AP3, AP4, and AP5), PCR-ribotyping, and PFGE (with SmaI endonuclease)-to differentiate 99 strains of C. difficile that had been previously serogrouped. Typing abilities were 100% for PCR-ribotyping and AP-PCR with AP3 and 90% for PFGE, due to early DNA degradation in strains from serogroup G. Reproducibilities were 100% for PCR-ribotyping and PFGE but only 88% for AP-PCR with AP3, 67% for AP-PCR with AP4, and 33% for AP-PCR with AP5. Discriminatory power for unrelated strains was >0.95 for all the methods but was lower for PCR-ribotyping among serogroups D and C. PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE. We conclude that PCR-ribotyping offers the best combination of advantages as an initial typing tool for C. difficile.

Bacterial Typing Techniques↗

PubMed2004

Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR.

BACKGROUND: Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as beta-actin (ACTB). RESULTS: The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired student's t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns. CONCLUSION: StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further, it correlates well with Taqman real time PCR in terms of quantitative and discriminatory ability. This label-free, inexpensive technique may provide the ability to generate prognostically important molecular signatures unique to individual tumors and may enable identification of novel therapeutic targets.

Binding, Competitive↗

PubMed2001

Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR.

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.

Animals↗

PubMed2001

Immunobead RT-PCR versus regular RT-PCR amplification of CEA mRNA in peripheral blood.

PURPOSE: The reverse transcription polymerase chain reaction (RT-PCR) amplification of tumor-specific mRNA has been used for the detection of cancer cells in peripheral blood. More recently, an immunomagnetic isolation and reverse transcription polymerase chain reaction (immunobead RT-PCR) was developed which has reportedly significant advantages over the previous RT-PCR analysis. In our study, we compared these two methods using a model set of peripheral blood containing tumor cells under standardized conditions. MATERIAL AND METHODS: In order to compare the false positive rate, normal peripheral blood samples from five volunteers were analyzed by both methods. A model set of peripheral blood containing tumor cells was established by adding SNUC4 human colon cancer cells to peripheral blood collected from normal volunteers not showing any nonspecific bands upon electrophoresis of the PCR products. RT-PCR amplification of carcinoembryonic antigen (CEA) mRNA was done with total RNA and mRNA prepared from this model sample. In immunobead RT-PCR analysis, mRNA was prepared from the cells isolated with anti-CEA antibody-coated magnetic beads or anti-Ber-EP4 antibody-coated magnetic beads before the RT-PCR analysis. RESULT: The immunobead RT-PCR yielded no non-specific band, while the regular RT-PCR using total RNA did show non-specific band formation in all five samples. When mRNA rather than total RNA was used, nonspecific bands were formed in three of the five samples. Immunobead RT-PCR allowed the detection of 10(1) tumor cells in 1 ml of peripheral blood. The regular RT-PCR analysis had a detection limit of 10(2) tumor cells in 1 ml of peripheral blood. CONCLUSION: The immunobead RT-PCR proved to be more sensitive and specific than the regular RT-PCR at least in our model system.

Antibodies, Monoclonal↗

PubMed2004

Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation.

BACKGROUND: Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) cause a wide range of signs and symptoms, varying from trivial mucocutaneous lesions to life-threatening infections, especially in immuno-suppressed patients. Since antiviral drugs are available, rapid and sensitive laboratory diagnosis of these virus infections is important. OBJECTIVE: To set up and evaluate HSV-1, HSV-2 and VZV qualitative real-time PCR on the Lightcycler system and to compare the results with those of the 'in-house' nested PCR and virus isolation. STUDY DESIGN: 110 consecutive samples from dermal or genital lesions from patients suspected of having HSV infections and another 110 samples from patients with suspected VZV infections were tested with real-time PCR, nested PCR and virus isolation. RESULTS: 24 samples (22%) were positive for HSV-1 by virus isolation and nested PCR, whereas 26 (24%) were positive by real-time PCR. HSV-2 was detected in 28 samples (25%) by virus isolation, in 41 (37%) by nested PCR and in 40 (36%) by real-time PCR. VZV was isolated in 15 samples (14%) and VZV DNA was detected in 51 samples (46%) by nested PCR as well as by real-time PCR. Nucleic acid amplification increased the detection rate of HSV-2 and VZV DNA in particular compared to virus isolation. No significant difference in sensitivity was found between real-time PCR and nested PCR. CONCLUSION: Real-time PCR has the advantage of rapid amplification, a reduced risk for contamination and it is a suitable method for diagnosis of VZV and HSV in specimens from skin lesions.

Chickenpox↗

PubMed1999

Quantitation of erbB-2 gene copy number in breast cancer by an improved polymerase chain reaction (PCR) technique, competitively differential PCR.

A new method of measuring gene copy number in small samples of DNA was used to measure amplification of the erbB-2 gene and a reference gene in breast cancers. The method, termed 'competitively differential polymerase chain reaction' (CD-PCR), combines the advantages of two other techniques for measuring amplification by PCR, namely differential PCR (D-PCR) and competitive PCR (C-PCR). The CD-PCR methodology was evaluated for sensitivity and specificity by comparing amplification measured by CD-PCR with that obtained by fluorescence in situ hybridization (FISH), C-PCR, and Southern blotting analysis. CD-PCR analysis proved to be an accurate predictor of amplification. CD-PCR also overcomes the problems involved in variation of PCR efficiencies and DNA concentrations in tumor samples, and the problems caused by the plateau effect in PCR.

Base Sequence↗

PubMed2001

Prospective BCR-ABL analysis by polymerase chain reaction (RT-PCR) in adult acute B-lineage lymphoblastic leukemia: reliability of RT-nested-PCR and comparison to cytogenetic data.

The reliability of routine BCR-ABL RT-nested-PCR was evaluated in 1453 B-lineage ALL or hybrid leukemia at initial diagnosis by RT-nested-PCR. All BCR-ABL-positive (n = 642) and 176 BCR-ABL-negative samples underwent a second RT-PCR. In 518 patients, karyotyping and/or FISH was compared to the BCR-ABL status. The second RT-PCR revealed in 155/642 initially positive samples a divergent result (153 BCR-ABL-negative, two other transcripts) that in most cases turned out to be caused by contaminations in the first RT-nested-PCR. Confirmatory RT-PCR detected 2/176 false negative first RT-nested-PCR results. Thirty-nine specimens remained ambiguous despite different RT-PCR approaches. As far as cytogenetic evaluation and FISH is available (n = 23), the majority but not all patients with an ambiguous RT-PCR result were Ph-negative (n = 18). RT-nested-PCR and cytogenetics yielded in 346 of 383 evaluable samples a concordant result. Differing results are given and account in part to the lower sensitivity of karyotyping. Taken together, confirmed RT-PCR detected BCR-ABL fusion transcripts consistently in 487 out of 1453 ALL samples (c-ALL: 43%, pre-B ALL: 34%, pro-B ALL: 5%, B-ALL: 0%, hybrid leukemia: 5/11). Since false positive initial RT-nested-PCR data were frequent, either confirmatory second RT-PCR or FISH analysis is warranted to guarantee sensitive and reliable results of utmost clinical relevance.

Adult↗

PubMed2002

Molecular typing of Helicobacter pylori by virulence-gene based multiplex PCR and RT-PCR analysis.

BACKGROUND: In recent years, numerous PCR amplification typing methods have been developed for the identification of H. pylori specific virulence genes. To reduce the number of PCR amplifications needed we have previously developed virulence gene based multiplex PCR and RT-PCR assays. AIM: The aim of the present study was to characterise Helicobacter pylori strains by means of virulence-gene based multiplex PCR and reverse-transcription PCR. SUBJECTS: Helicobacter pylori clinical reference strains HP-HJM 1-25 originally obtained from routine cultures of clinical gastric biopsy specimens from dyspeptic patients were used in the present study. METHODS: Helicobacter pylori multiplex PCR and RT-PCR assays were carried out using primer pairs targeting the cagA, vacA, ureA, ureI, hspA (hsp60), and sodB genes and their cDNA. RESULTS: Unexpected multiplex PCR and RT-PCR patterns were observed by ethidium bromide staining of agarose gels and Southern blot hybridisation analysis. DNA sequence analysis revealed a complex pattern of point mutations, deletions and insertions in the sodB, cagA and vacA genes, respectively. Mutations occurring in the PCR and hybridisation primer binding sites might explain some of the discrepancies previously observed in the expression of these genes. Furthermore, the present data show that coexpression of cagA and vacA transcripts of different sizes may take place in the same strain. CONCLUSIONS: The multiplex PCR and RT-PCR assay described allows rapid characterisation of H. pylori virulence genes at the DNA and RNA (cDNA) levels. However, extensive DNA sequence analysis seems necessary if one wants to reveal details of mutations occurring in the cagA and vacA genes.

Amino Acid Sequence↗