Single-round Polymerase Chain Reaction Research Articles

Hepatitis C subtyping assay failure in UK patients born in sub-Saharan Africa: Implications for global treatment and elimination.

HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction(PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools. Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis. This study provides a simple low-cost pathway to accurately subtype in SSA, guide regional therapeutic choice and assist global surveillance and elimination initiatives.

High molecular prevalence of HPV and other sexually transmitted infections in a population of asymptomatic women who work or study at a Brazilian university

ABSTRACTSexually transmitted infections (STIs) represent a global health problem with variable prevalence depending on the geographical region and the type of population. Human papillomavirus (HPV) encompasses widespread virus types related to cervical carcinogenesis. The present study investigated the molecular prevalence of HPV and seven other important STIs in asymptomatic women working or studying at a Brazilian university. A secondary aim was to assess cytological abnormalities associated with HPV and other STIs coinfections. We recruited 210 women from a Brazilian university. HPV was detected using a single-round polymerase chain reaction (sPCR) followed by a viral genotyping by restriction fragment length polymorphism (RFLP-PCR). The presence of seven STIs: Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, herpes simplex virus (HSV)-1 and HSV-2 was detected by multiplex PCR (M-PCR). Furthermore, cytological findings and epidemiological characteristics were evaluated.The mean age of the participants was 27.1 years old. HPV prevalence was 33.8%, and HPV16 was the most frequently detected papillomavirus genotype. Moreover, multiple HPV infections were common (42.2%). We detected at least one STI agent in 11.4% of the tested women, most frequently C. trachomatis (6.7%). Among HPV-positive women, 14.1% were coinfected with other STI agents. Cytological abnormalities were observed in 9.5% of smears, and HPV-DNA, high-risk HPV (HR-HPV), HPV16 and HPV multiple infections were associated with abnormal cytological findings. There was a high prevalence of HPV, and C. trachomatis was the most prevalent STI agent, with low rates of cytological abnormalities. These findings highlight the need of timely STI diagnosis in young asymptomatic women and of a public policy design for STI prevention.

Porcine lymphotropic herpesvirus (Gammaherpesvirinae) DNA in free-living wild boars (Sus scrofa Linnaeus, 1758) in Brazil.

BackgroundSuid gammaherpesvirus 3, 4, and 5 (porcine lymphotropic herpesvirus – PLHV-1, -2, and -3) are viruses that infect domestic and feral pigs.ObjectivesThis study examined the presence of PLHV DNA in biological samples from free-living wild boars circulating in a Brazilian geographical region with a high density of commercial domestic pigs.MethodsLung samples of 50 free-living wild boars were collected by exotic wildlife controller agents between 2017 and 2019 in the state of Paraná, southern Brazil. Lung and spleen fragments were obtained from six fetuses collected by hysterectomy post mortem from a pregnant sow. A polymerase chain reaction (PCR) assay using consensus primers (pan-herpesviruses) was performed to detect PLHV DNA. The samples showing positive results for PLHV DNA were submitted to single-round PCR assays with the specific primers for identifying PLHV-1 (213-S/215-As), PLHV-2 (208-S/212-As), and PLHV-3 (886s/886As). The specificity of the species-specific PCR products was assessed by nucleotide sequencing of the amplicons.ResultsForty-eight (96%) of the 50 lung samples analyzed were positive for PLHV by PCR using pan-herpesvirus primers. In 33 (68.75%) of the positive samples, at least two PLHV species were identified simultaneously. The DNA of PLHV-1, -2, and -3 was found in free-living wild boars of all ages, but not in the fetuses, even though they were from a sow that tested positive for all three viruses.ConclusionThese viruses are endemic to the population of feral pigs in the Brazilian region evaluated, as well as in domesticated pigs.

Comparison of Direct Examination and In Vitro Culture for the Detection of Blastocystis spp. in Orang Asli Stool Samples

Microscopy-based technique has been widely used in the detection of Blastocystis sp. This study was conducted to compare the techniques used for screening of Blastocystis sp., namely in vitro cultivation of stool specimens in Jones’ medium (IVC) followed by Wheatley Trichrome staining and direct examination of stool samples preserved with polyvinyl alcohol (PVA) followed by Wheatley Trichrome staining with single-round polymerase chain reaction (PCR) as the reference technique. The study was performed on 466 stool samples obtained from the aboriginal community in Pahang, Malaysia. IVC showed higher detection rate of Blastocystis sp. (35.6%) than PVA (20.0%). Single-round PCR detected Blastocystis sp. in 41.0% of the stool specimens. The sensitivity and specificity of PVA and IVC in comparison to the reference technique were 75.3% (95% CI: 65.2-83.6) and 68.5% (CI: 63.7-73.3) and 88.6% (CI: 82.7-93.0) and 86.3% (CI: 81.9-90.0), respectively. The agreement between the reference technique and PVA showed statistically significant fair agreement by Cohen Kappa statistics of (K=0.318, p<0.001), meanwhile statistically significant substantial agreement was observed between PCR and IVC by Cohen Kappa (K=0.727, p<0.001). Therefore, in vitro cultivation in Jones’ medium followed by Wheatley Trichrome staining of stool specimens should be used as a screening technique in the detection of Blastocystis sp. infections.

A Novel Oligonucleotide Pair for Genotyping Members of the Pseudomonas Genus by Single-Round PCR Amplification of the gyrB Gene

Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species.

English

Malaria is still a public health problem in the world. It accounted for an estimated 214 million cases and 438,000 deaths in the year 2015. During the dry season, most people are likely to be asymptomatic and therefore fail to be diagnosed with malaria. This study established the proportion of people who came to health facility, escorting sick relatives and had detectable malaria parasites. This was a cross sectional study. All relatives who escorted sick patients to Karume Health Centre between August and December 2013 were screened for malaria using malaria rapid diagnostic test (mRDT) and single round PCR targeting mitochondrial DNA. A total of 400 relatives were screened for malaria using two methods. Prevalence of malaria was 14.5 and 16.8% by mRDT and polymerase chain reaction (PCR), respectively. The prevalence of malaria was higher among febrile patients by methods, mRDT (17.8%) and PCR (17.1%), respectively. The prevalence of asymptomatic malaria was 16.4 and 16.5% by mRDT and PCR, respectively. The overall agreement between the two tests was 87.1% with positive agreement of 63.8% and negative agreement of 91.2%. There were a substantial proportion of patients with malaria who visited the health facilities during the dry season. mRDT and single round PCR targeting mitochondrial DNA had a good agreement and can be used for detection of both symptomatic and asymptomatic malaria. Provider initiated screening can help to improve malaria detection during the dry season, as we move towards reduced malaria prevalence and elimination phase. Key words: Asymptomatic malaria, prevalence of malaria among relatives, mitochondrial DNA, polymerase chain reaction (PCR).

Cultivation of Entamoeba species in Lock’s -egg medium and differentiation of E.moshkoviskii ,E.dispar and Ehistolytica by using single round polymerase chain reaction

The genus Entamoeba contains many species, three of which are identical (Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii ) reside in the human intestinal lumen. Entamoeba histolytica is the causative agent of amebiasis and mortality worldwide in humans. Although recent studies highlight the recovery of E. dispar andE. moshkovskii from patients with gastrointestinal symptoms. The cultivation of three identical Entamoeba species was done successfully in Lock’s-egg medium , a high number of trophozoite were found in culture .these three identical Entamoeba species did not differentiation by using microscope .but the devolution of scientific they could differentiation by using PCR technique when extraction the DNA in stool samples by using specific bands and primers

Occurrence of Blastocystis sp. in water catchments at Malay villages and Aboriginal settlement during wet and dry seasons in Peninsular Malaysia.

In the tropics, there are too few studies on isolation of Blastocystis sp. subtypes from water sources; in addition, there is also an absence of reported studies on the occurrence of Blastocystis sp. subtypes in water during different seasons. Therefore, this study was aimed to determine the occurrence of Blastocystis sp. subtypes in river water and other water sources that drained aboriginal vicinity of highly endemic intestinal parasitic infections during wet and dry seasons. Water samples were collected from six sampling points of Sungai Krau (K1–K6) and a point at Sungai Lompat (K7) and other water sources around the aboriginal villages. The water samples were collected during both seasons, wet and dry seasons. Filtration of the water samples were carried out using a flatbed membrane filtration system. The extracted DNA from concentrated water sediment was subjected to single round polymerase chain reaction and positive PCR products were subjected to sequencing. All samples were also subjected to filtration and cultured on membrane lactose glucuronide agar for the detection of faecal coliforms. During wet season, Blastocystis sp. ST1, ST2 and ST3 were detected in river water samples. Blastocystis sp. ST3 occurrence was sustained in the river water samples during dry season. However Blastocystis sp. ST1 and ST2 were absent during dry season. Water samples collected from various water sources showed contaminations of Blastocystis sp. ST1, ST2, ST3 and ST4, during wet season and Blastocystis sp. ST1, ST3, ST8 and ST10 during dry season. Water collected from all river sampling points during both seasons showed growth of Escherichia coli and Enterobacter aerogenes, indicating faecal contamination. In this study, Blastocystis sp. ST3 is suggested as the most robust and resistant subtype able to survive in any adverse environmental condition. Restriction and control of human and animal faecal contaminations to the river and other water sources shall prevent the transmission of Blastocystis sp. to humans and animals in this aboriginal community.

An improved single-round PCR leads to rapid and highly sensitive detection of Pneumocystis spp.

In order to standardize a polymerase chain reaction (PCR)-based method of Pneumocystis detection, we describe the development of an improved PCR method that targets the Pneumocystis mtLSUrRNA gene. Design of a new primer pair and PCR program with suitable parameters and optimization resulted in a simpler and faster single-round amplification assay. The sensitivity of the novel Pneumocystis genus-specific PCR proved comparable to the reference nested PCR. The improvement that this new PCR assay offers in the detection and epidemiological studies of Pneumocystis spp. infection in research laboratories is discussed.

Outbreak of Pneumocystis Pneumonia in Renal and Liver Transplant Patients Caused by Genotypically Distinct Strains of Pneumocystis jirovecii

An outbreak of 29 cases of Pneumocystis jirovecii pneumonia (PCP) occurred among renal and liver transplant recipients (RTR and LTR) in the largest Danish transplantation centre between 2007 and 2010, when routine PCP prophylaxis was not used. P. jirovecii isolates from 22 transplant cases, 2 colonized RTRs, and 19 Pneumocystis control samples were genotyped by restriction fragment length polymorphism and multilocus sequence typing analysis. Contact tracing was used to investigate transmission. Potential risk factors were compared between PCP cases and matched non-PCP transplant patients. Three unique Pneumocystis genotypes were shared among 19 of the RTRs, LTRs, and a colonized RTR in three distinct clusters, two of which overlapped temporally. In contrast, Pneumocystis control samples harbored a wide range of genotypes. Evidence of possible nosocomial transmission was observed. Among several potential risk factors, only cytomegalovirus viremia was consistently associated with PCP (P=0.03; P=0.009). Mycophenolate mofetil was associated with PCP risk only in the RTR population (P=0.04). We identified three large groups infected with unique strains of Pneumocystis and provide evidence of an outbreak profile and nosocomial transmission. LTRs may be infected in PCP outbreaks simultaneously with RTRs and by the same strains, most likely by interhuman transmission. Patients are at risk several years after transplantation, but the risk is highest during the first 6 months after transplantation. Because patients at risk cannot be identified clinically and outbreaks cannot be predicted, 6 months of PCP chemoprophylaxis should be considered for all RTRs and LTRs.

Single step detection of HIV-1 proviral DNA and housekeeping β-actin gene from dried blood spots by a monoplex polymerase chain reaction

There is a growing need for developing a simple, rapid, reliable and cost effective method for detection of HIV-1 for early diagnosis of the infection especially in developing countries and in resource limited settings. A method for simultaneous detection of the HIV-1 p17 gene and the house keeping human β-actin gene from dried blood spots (DBS) by a monoplex polymerase chain reaction (PCR) is described. Genomic DNA was extracted from 40 HIV-1 positive and 40 HIV-1 negative DBS and used as templates to amplify both the HIV-1 p17 and β-actin genes simultaneously under the same cycling condition by a single round PCR. This method of detection of HIV-1 may provide a simple, rapid and cost effective alternative in resource limited settings; however, it would require testing a larger number of samples before widespread use.

Discrimination Between Human and Animal DNA

Identification of a report's species is one of the basic analyses in forensic laboratories. The authors report the case of 6 bone fragments recovered in a wooded area, which were not attributable to 1 animal species on the basis of morphologic examination. The aim of this study was to develop a duplex polymerase chain reaction (PCR) to discriminate human and animal origin of bone fragments. The method is based on the PCR amplification of cytochrome b and a 16S ribosomal mitochondrial DNA fragment, which has never been tested up to now. Our protocol combines a single-round PCR with direct visualization of amplicons in agarose gel, without sequencing analysis of the PCR products. The presence of a single band (359 bp) indicates a nonhuman origin of the sample, whereas 2 bands (157 and 359 bp) indicate a human biologic sample.This method revealed to be useful for forensic purposes because the 16S ribosomal mitochondrial DNA is a small human-specific fragment that is easily amplifiable even with degraded DNA from biologic materials such as old bones.

Merkel Cell Polyomavirus Infection in HIV-Positive Men

To evaluate Merkel cell polyomavirus (MCPyV) DNA prevalence and load among men with human immunodeficiency virus (HIV) (hereafter referred to as HIV-positive men) and among healthy male control subjects. Prospective study from February 4, 2009, through April 24, 2010. Dermatology department of a university hospital. A total of 449 male adults were prospectively recruited, including 210 HIV-positive men who have sex with men and 239 healthy controls. Cutaneous swabs were obtained once from the surface of the forehead in all participants. Swabs were evaluated for the presence of MCPyV DNA using single-round and nested polymerase chain reaction. The MCPyV DNA load (the number of MCPyV DNA copies per β-globin gene copy) was determined in MCPyV-positive samples using quantitative real-time polymerase chain reaction. Among 449 forehead swabs analyzed, MCPyV DNA was detected in 242 (53.9%). Compared with healthy controls, HIV-positive men more frequently had MCPyV DNA on nested polymerase chain reaction (49.4% vs 59.0%, P = .046) and on single-round polymerase chain reaction (15.9% vs 28.1%, P = .002). The MCPyV DNA loads in HIV-positive men were similar to those in HIV-negative men, but HIV-positive men with poorly controlled HIV infection had significantly higher MCPyV DNA loads than those who had well-controlled HIV infection (median and mean MCPyV DNA loads, 2.48 and 273.04 vs 0.48 and 11.84; P = .046). Cutaneous MCPyV prevalence is increased among HIV-positive men who have sex with men. Furthermore, MCPyV DNA loads are significantly higher in HIV-positive men with poorly controlled HIV infection compared with those who have well-controlled HIV infection. This could explain the increased risk of MCPyV-associated Merkel cell carcinoma observed among HIV-positive individuals.

Evaluation of a single round polymerase chain reaction assay using dried blood spots for diagnosis of HIV-1 infection in infants in an African setting

BackgroundThe aim of this study was to develop an economical 'in-house' single round polymerase chain reaction (PCR) assay using filter paper-dried blood spots (FP-DBS) for early infant HIV-1 diagnosis and to evaluate its performance in an African setting.MethodsAn 'in-house' single round PCR assay that targets conserved regions in the HIV-1 polymerase (pol) gene was validated for use with FP-DBS; first we validated this assay using FP-DBS spiked with cell standards of known HIV-1 copy numbers. Next, we validated the assay by testing the archived FP-DBS (N = 115) from infants of known HIV-1 infection status. Subsequently this 'in-house' HIV-1 pol PCR FP-DBS assay was then established in Nairobi, Kenya for further evaluation on freshly collected FP-DBS (N = 186) from infants, and compared with findings from a reference laboratory using the Roche Amplicor® HIV-1 DNA Test, version 1.5 assay.ResultsThe HIV-1 pol PCR FP-DBS assay could detect one HIV-1 proviral copy in 38.7% of tests, 2 copies in 46.9% of tests, 5 copies in 72.5% of tests and 10 copies in 98.1% of tests performed with spiked samples. Using the archived FP-DBS samples from infants of known infection status, this assay was 92.8% sensitive and 98.3% specific for HIV-1 infant diagnosis. Using 186 FP-DBS collected from infants recently defined as HIV-1 positive using the commercially available Roche Amplicor v1.5 assay, 178 FP-DBS tested positive by this 'in-house' single-round HIV-1 pol PCR FP-DBS PCR assay. Upon subsequent retesting, the 8 infant FP-DBS samples that were discordant were confirmed as HIV-1 negative by both assays using a second blood sample.ConclusionsHIV-1 was detected with high sensitivity and specificity using both archived and more recently collected samples. This suggests that this 'in-house' HIV-1 pol FP-DBS PCR assay can provide an alternative cost-effective, reliable and rapid method for early detection of HIV-1 infection in infants.

Detection of Cyprinid Herpesvirus-3 in Field Samples of Common and Koi Carp by Various Single-Round and Nested PCR Methods

Cyprinid herpesvirus-3 (CyHV-3, previously described as koi herpesvirus) is a causative agent of the lethal disease in koi and common carp that can adversely affect common and koi carp breeds. Outbreaks with mass mortality have occurred world wide since 1998. At present, several different polymerase chain reaction (PCR) tests are in use to detect CyHV-3 in koi and common carp. In this study, we compared the sensitivity of the single-round PCR assays of Gray, Gilad, and Bercovier, and further the Gilad and Bercovier-nested PCR assays by testing tissue samples with known amounts of CyHV-3 DNA and by testing the panel of field tissue samples (106) of koi and common carp. The sensitivity of all the PCR protocols used in this study was similar within testing the spiked homogenate sample with a known amount of CyHV-3 DNA (ca. 12 DNA copies), with the exception of Gray PCR (25 copies). Detection levels of the individual PCR methods in field samples varied. The single-round Bercovier method was the most sensitive in comparison with the following two single-round methods, based on the number of positive field samples (34%). The improvement of sensitivity and confirmation of specifity were sufficient when the two-round Bercovier method was used with a detection of 44.3% of positive samples in total.

Diagnostic significance of nested polymerase chain reaction for sensitive detection of Pneumocystis jirovecii in respiratory clinical specimens

A total of 327 clinical specimens, including both invasive and noninvasive samples, obtained from 275 patients with various types of underlying immunocompromised conditions and a clinical suspicion of Pneumocystis pneumonia (PCP) were subjected to 2 different nested polymerase chain reaction (PCR) assays. The target genes used for nested PCR were mitochondrial large subunit ribosomal RNA (mtLSU rRNA) and internal transcribed spacer (ITS) region. The results were compared with a single-round PCR targeting major surface glycoprotein (MSG) gene. Amplification was successful in 16% of cases by mtLSU rRNA nested PCR, in 14.5% by ITS nested PCR, and in 10.9% by MSG PCR. The nested mtLSU rRNA PCR was found to be more sensitive (100% sensitive and 98.7% specific) and useful in detecting PCP for its use in routine diagnosis in our settings. Thus, this assay may be quite useful in the identification of patients who are in the early stage of Pneumocystis jirovecii infection with an organism load that could not be easily detected by the single-step PCR.

Rapid Amplification of Flanking Sequences of a Known DNA Region by Partial Restriction Digestion and Hot Start PCR

A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single round polymerase chain reaction. Total genomic DNA was partially digested with the frequent-cutting restriction enzyme Mse I. The partially digested products were ligated to an unphosphorylated adaptor. A hot start PCR amplification with Ta q polymerase and dNTP was performed with a DNA-specific primer and the adaptor primer complementary to the adaptor and the Mse I recognition site. The amplified products were fractionated, cloned and sequenced. By this method, we cloned the downstream region of a gynoecious marker TG/CAC 234 from cucumber ( Cucumis sativus L.).

Evaluation of molecular techniques in prediction and diagnosis of cytomegalovirus disease in immunocompromised patients

OBJECTIVES: To evaluate selected molecular tests in diagnosis and screening of cytomegalovirus (CMV) infection in immunosuppressed patients. DESIGN: Clinical and cost-effectiveness were assessed through a prospective two-stage trial of CMV screening regimes in a routine service setting. Different molecular test results were fed back to clinicians in each stage, plus antigenaemia results. The technical performance of the molecular methods was assessed through an independent masked comparison of each molecular test against the established (antigenaemia) test. Scientists performing a particular test were blind to the other test results for that sample. Diagnostic and therapeutic impact were recorded prospectively for all tests, to include any effect on diagnostic certainty, changes to CMV therapy and any other reported impact on patient management. The cost of each test was estimated under different laboratory conditions. Prospective patients undergoing CMV screening were compared with consecutive historical controls in the same unit. Towards the end of the study, a survey of all UK virology laboratories was undertaken to identify current CMV screening practice and test preferences. In addition, all UK renal transplant surgeons and haematology transplant centres were surveyed in order to identify current clinical practice and perceptions of the benefits of CMV screening. SETTING: Study patients were recruited from University Hospital Wales (UHW), Cardiff. Staff in the Cardiff Public Health Laboratory Service virology laboratory performed the tests. PARTICIPANTS: A consecutive series of transplant patients was recruited to the prospective study over a 42-month period, totalling 98 renal and 140 haematology patients. A consecutive series of historical controls was identified, with 199 renal and 136 haematology patients who underwent transplants in the UHW during the 29 months prior to the prospective CMV screening trial.

Evaluation of quantitative real-time PCR for rapid assessments of the exposure of sentinel fish to Myxobolus cerebralis

Pathogen-free rainbow trout (Oncorhynchus mykiss) aged 735 degree days were experimentally exposed to a low dose of infectious Myxobolus cerebralis (20 triactinomyxons fish(-1)). Three time periods were chosen for sampling that included 10 days (d), 67 d, and 5 months (mo) post exposure. Five diagnostic assays were used: (1) conventional single-round polymerase chain reaction (PCR), (2) nested PCR, (3) real-time TaqMan PCR, (4) pepsin-trypsin digest, and (5) histopathology. M. cerebralis was detected among individual rainbow trout by all of the PCR diagnostic tests employed at each of the three sampling time points. This result demonstrates that any of these three diagnostic approaches are capable of detecting the parasite from infected fish tissues under the conditions tested. Real-time PCR provided good biological evidence that parasite replication increases temporally as shown by quantification values that were significantly different (P<0.0001) at 10 d as compared to 67 d and 5 mo postexposure. Although sampling at 10 d by real-time PCR may be too early to accurately predict quantities of the parasite that will be present at 5 mo, it does forecast the proportions of fish that are likely to be infected at 67 d and 5 mo postparasite exposure. Real-time PCR could potentially be used as a quantitative diagnostic PCR tool to predict parasite load and outcome of M. cerebralis infection.

Detection of BCL2-IGH Using Single-Round PCR Assays

The translocation t(14;18) resulting in fusion of the BCL2 and the immunoglobulin heavy chain genes (BCL2-IGH) is present in 80% to 90% of follicular lymphomas and 20% to 30% of diffuse large B-cell lymphomas. Polymerase chain reaction (PCR) analysis for the translocation products suffers from low analytic specificity. As a result, either nested PCR or probe hybridization is required to aid in the identification of the specific translocation products. These added procedures are undesirable in clinical laboratories because nested procedures increase the possibility of contamination and probe hybridization increases assay turnaround time. To simplify the BCL2-IGH assay procedure, we attempted to eliminate the nonspecific PCR products by optimizing the annealing temperatures of the PCR assays using a gradient thermocycler. We showed that gradually increasing the annealing temperature from 55 degrees C to 67 degrees C significantly enhanced the intensity of the specific PCR products while eliminating the nonspecific ones. We compared the simplified procedure with a PCR-probe hybridization method on 68 patient specimens. The simplified procedure had increased analytic and diagnostic specificities with comparable sensitivities. With significantly improved analytic specificity, one round of PCR is sufficient to detect the BCL2-IGH gene rearrangements without further confirmation.