Single Polymerase Chain Reaction Assay Research Articles

A one-step multiplex PCR assay for the detection and differentiation of four species of Clarireedia causing dollar spot on turfgrass.

Dollar spot (DS) is one of the most destructive and economically important diseases of cool- and warm-season turfgrasses worldwide. A total of six species causing DS disease in the genus Clarireedia have been described, and four of them have been reported to be distributed countrywide in China. Identification of different species of Clarireedia is a prerequisite for the effective management of DS disease. Here we report a novel polymerase chain reaction (PCR)-based method for the detection and differentiation of the four species of Clarireedia associated with DS on turfgrass in China: C.jacksonii, C.paspali, C.monteithiana and C.hainanense. Species-specific genes were identified for each species by comparative genomics analysis. Four primer pairs were designed and mixed to amplify species-specific PCR fragments with differential sizes for the four species of Clarireedia in a single multiplex PCR assay. No PCR products were generated from the DNA templates of other common fungal pathogens associated with multiple turfgrass diseases. The multiplex PCR method developed can be used for the rapid and accurate detection and differentiation of the four species of Clarireedia from pure cultures as well as from infected turfgrass blades with DS symptoms. The study developed a one-step multiplex PCR assay for the detection and differentiation of four species of Clarireedia causing DS on turfgrass in China, which will have important implications for DS management in China and worldwide. © 2022 Society of Chemical Industry.

A new single run polymerase chain reaction assay for cyclosporiasis in immunocompromised patients

causes human intestinal cyclosporiasis. It is more common in the immunocompromised patients and mainly seen in people living with HIV/AIDS (PLHA), post-renal transplant (PRT) patients and immunocompromised children (IC). Diagnostic microscopy for the oocysts of the parasite is less sensitive, requiring examination of multiple stool samples. Here we developed a new single run polymerase chain reaction (PCR) assay for the detection of and it was used to know the hospital based prevalence of cyclosporiasis. A cross-sectional study was conducted from June 2016 to October 2020 in a tertiary care teaching hospital. A new single run amplification PCR-based diagnostic assay was developed for . Stool samples were collected from 121 PLHA, 135 PRT and 79 immunocompromised children (IC) other than PLHA and PRT. All stool samples were examined for the presence of oocysts as well as tested with new PCR assay. Modified Ziehl-Neelsen staining of the concentrated stool smear did not reveal oocysts of species in any stool specimen. However, new PCR assay detected in 2 stool specimens – one from a PLHA patient and another from a PRT patient, giving a prevalence of 0.6% (2/335), 0.8% (1/121) in PLHA and 0.7% (1/135) in PRT. It was not detected in IC. Cyclosporiasis is infrequent in southern part of India. The new single run PCR assay developed by us is simple and cost effective molecular assay for the detection of .

DD Genotype and Atherosclerosis in Overweight Menopausal Women

Abstract Background Sex-specific pathology of coronary artery disease (CAD) has not been recognized. Women with obstructive or nonobstructive CAD associated with traditional risk factors have similar events; no studies have explored both populations in association with genetic markers. Objective To evaluate the DD genotype in overweight menopausal women and its association with CAD and traditional risk factors. Method This cross-sectional study included 356 menopausal women who underwent coronary angiography as CAD assessment. The patients’ DNA was extracted and polymorphisms were detected with a single polymerase chain reaction assay. Two groups were formed based on luminal lesions (normal [n = 134] or pathological [n = 222]) with a cutoff value > 30%, considering overweight and age. The chi-square test, Student’s t-test, and multivariate logistic regression were performed as appropriate (p 25 was considered (33% vs. 18%, p = 0.012). In multivariate analysis with two threshold levels (> 50 and > 60 years), diabetes was significantly associated with CAD in both models (p = 0.021 vs. 0.009) but the genotype was only associated with younger age (p = 0.034). Conclusion These data support an association between atherosclerosis and the renin–angiotensin system in overweight menopausal women that is dependent on the age at which the ischemic event occurs.

Fecal Colonization of Extended-Spectrum Beta Lactamase-Producing Salmonella spp. in Broilers in Lorestan Province of Iran

Background: Poultry is considered as a major source of human contamination with nontyphoidal Salmonella species. Global concern regarding the emergence and dispersion of extended-spectrum beta-lactamase (ESBLs)-producing isolates in broilers has increased during recent years. Objective: This study was proposed to evaluate the prevalence of Salmonella and the associated ESBLs in broilers in Lorestan province of Iran. Materials and Methods: Five hundred fresh fecal samples of broilers were phenotypically screened for Salmonella. The isolates were confirmed molecularly using an invA-based polymerase chain reaction (PCR). Confirmatory combination disk method was applied for phenotypic detection of ESBLs among the isolates, followed by molecular identification of blaCTX-M, blaTEM, and blaSHV genes in 3 single PCR assays among positive isolates. Chi-square test in SPSS software was used for the assessment of statistical relationships. Results: Of the 95 Salmonella isolates detected using routine bacteriological methods, all were confirmed molecularly. They generated the expected 254-bp amplicon. Moreover, 13 isolates were phenotypically recognized as ESBL determinants, among which 9 and 4 harbored blaCTX-M and blaTEM, respectively. No blaSHV and co-existence of the genes were determined. Conclusion: The threat imposed by dissemination of ESBL-producing non-typhoidal Salmonella spp. isolated from broilers was confirmed in the studied region. Continuous monitoring programs, application of biosecurity measures, and prudent prescription of antibiotics are warranted in order to prevent the introduction or dispersion of the ESBL-producing Salmonella.

Development of a Single Reaction PCR Genotyping Assay for Two Separate Gene Loci in Mice

There are many variables to consider when optimizing a polymerase chainreaction (PCR) assay for a specific gene—from methods of DNA extraction to choosing an effective combination of primers. A multiplex PCR assay analyzing two genes in the same reaction may require additional optimization. The objective of this experiment was to optimize a single PCR assay to effectively genotype C57BL/6 transgenic mouse strains in which two genes have been modified. By removing a portion of the Es1 gene, the expression of serum carboxylesterase was interrupted, resulting in serum carboxylesterase knock out (Es1 KO) mice. In another location, the gene expressing mouse acetylcholinesterase was replaced by a gene expressing human acetylcholinesterase, resulting in a human acetylcholinesterase knock in (AChEKI) mouse strain. There are several possible genotypes for each mouse strain: for Es1 KO mice the possibilities are wild type (WT +/+), knock out (KO −/−), or heterozygous (Het +/−), and for AChEKI mice the possibilities are human (H/H), mouse (M/M), or heterozygous (M/H). Optimization of the PCR genotyping assays for these strains was achieved by altering the method of DNA extraction, annealing temperatures, and testing multiple primer sets. Initially, DNA was extracted from mouse fecal matter, but this method was later determined to produce unreliable results in the subsequent PCR assays. DNA extracted from tail tissue was found to be much more reliable. Utilizing tail tissue DNA, a single pair of primers was found that could differentiate WT, KO, and Het alleles in the Es1 KO strain. The optimal annealing temperature for these primers was determined using a temperature gradient experiment. Primers for genotyping the AChE KI strain were selected and optimized in a similar manner. Finally, a multiplex PCR assay was developed allowing for the simultaneous determination of the genotype of both genes in mice.Support or Funding Information*This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U. S. Army Medical Research and Materiel Command.The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and the Animal Welfare Act of 1966 (P.L. 89‐544), as amended.Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.The views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiplex quantification of Escherichia coli, Salmonella typhi and Vibrio cholera with three DNA targets in single reaction assay

Escherichia coli (E. coli), Salmonella typhi and Vibrio cholera harmful pathogens, which causes various diseases in humans. Rapid diagnosis of bacterial infection is an important for patient management and appropriate therapy during the early phase of the bacterial infected diseases. Among the existing techniques for identifying pathogens were less sensitive and time-consuming processes. In the present study total, 48 clinical 31 blood and 17 urine samples of patients suspected with the infections were collected from SVRR Hospital and used to detect the pathogens. Multiplex polymerase chain reaction (PCR) assay was set to design for the identification of Escherichia coli, Salmonella typhi and Vibrio cholera from the different clinical samples. Rapid diagnosis of Escherichia coli (E. coli), Salmonella and Vibrio cholera pathogens can be done with simultaneously in a single multiplex PCR assay by using specific primers with adjusted PCR conditions. Through this approach, the results represented with out of 31 blood samples 1–15 shows the positive with E. coli and remaining 14 only 11 were correlated with multiplex results of Vibrio cholera, remaining the urine samples all are positive with 17 samples correlate with the Salmonella typhi. Through the high specificity benefits of excellent sensitivity, with high resolution and reproducibility. This method of results proved and illustrates the best potential system for diagnosing the infectious disease with modern trendy.

Use of Hybrid Rice and the Proliferation of Weedy Rice: A Case in Jiangsu, China

Jiangsu Province, located between northern China and southern China, is a typically mixed area of conventional and three‐line hybrid rice without Oryza rufipogon Griff. and Oryza nivara S. D. Sharma & Shastry. In the recent decade, occurrence of weedy rice rapidly increased year by year since it was found and reported from 2000. The recent cloning of the CMS‐WA gene (wild abortive cytoplasmic male sterility) makes it possible to design a marker specific for the gene for testing large quantities of weedy rice samples in a single assay by polymerase chain reaction (PCR). Through performing a BLAST search in the National Center for Biotechnology Information (NCBI) database using the sequence of the CMS‐WA gene cloned from ZS97A as a reference, the major difference between three‐line hybrid rice and conventional rice cultivars was found in the 287 to 439 bp region of the CMS‐WA gene. A specific marker, WA7, was designed and evaluated for this region of the CMS‐WA gene. This marker, in combination with RED4, a specific marker for the Rc red pericarp gene, was used to distinguish weedy rice, three‐line hybrid rice, and conventional rice in a single PCR assay. A total of 1232 accessions of weedy rice collected from 63 locations in Jiangsu Province, China, were evaluated using the red pericarp and CMS‐WA DNA markers. Only four accessions from three locations were found positive for the CMS‐WA gene, accounting for just 4.76 and 0.32% of all locations and accessions, respectively. These results indicate that the three‐line hybrid is likely a source but not the major source of weedy rice in Jiangsu Province.

Utility of a single nasal polymerase chain reaction assay in predicting absence of skin and environmental contamination in hospitalized patients with past methicillin-resistant Staphylococcus aureus

We evaluated hospitalized patients with a history of methicillin-resistant Staphylococcus aureus (MRSA) for persistent colonization and need for contact precautions. Up to 3 daily cultures of nares, skin, and any present wounds were compared with a single nasal polymerase chain reaction (PCR) assay. Most patients (76.2%) were no longer colonized with MRSA. A single PCR assay was sufficient to exclude persistent colonization and environmental contamination and remove the contact precautions.

Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections

Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections.

Development of PCR-Based Assays for Detecting and Differentiating Three Species of Botrytis Infecting Broad Bean.

Botrytis cinerea, B. fabae, and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop polymerase chain reaction (PCR)-based assays to detect and differentiate these three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, were designed based on two sequence-characterized amplified region markers derived from two random amplified polymorphic DNA assays. The other primer set, Bfa-f/Bfa-r for B. fabae, was designed based on the necrosis and ethylene-inducing protein 1 gene sequence. The three primer sets were highly specific for the corresponding species of Botrytis in both single and multiplex PCR assays. The PCR detection limit was 40, 40, and 400 pg of DNA per 25-μl reaction mixture for B. fabae, B. fabiopsis, and B. cinerea, respectively. Presence of the broad bean DNA in the PCR reactions at 1:1000 (Botrytis DNA/broad bean DNA [wt/wt]) had negligible effects on detection of the targeted Botrytis spp. The multiplex PCR assay was able to detect three Botrytis spp. in artificially infected and naturally infected broad bean leaves. These results suggest that the multiplex PCR assay developed in this study could be used to monitor the epidemics of chocolate spot of broad bean in the field.

Development of a novel single tube nested PCR for enhanced detection of cytomegalovirus DNA from dried blood spots

Newborn screening for congenital cytomegalovirus (CCMV) using dried blood spots (DBS) has been proposed because many developed countries have DBS screening programmes in place for other diseases. The aim of this study was to develop a rapid, single tube nested polymerase chain reaction (PCR) method for enhanced detection of CMV from DBS compared to existing (single target) real time PCRs. The new method was compared with existing real time PCRs for sensitivity and specificity. Overall sensitivity of the single target PCR assays in both asymptomatic and symptomatic infants with laboratory confirmed congenital CMV was 69% (CMV PCR or culture positive before day 21 of life). In contrast, the single tube nested assay had an increased sensitivity of 81% with100% specificity. Overall the assay detected CMV from a DBS equivalent to an original blood sample which contained 500IU/ml. In conclusion this single tube nested methodology allows simultaneous amplification and detection of CMV DNA in 1.5h removing the associated contamination risk of a two step nested PCR. Owing to its increased sensitivity, it has the potential to be used as a screening assay and ultimately allow early identification and intervention for children with congenital CMV.

Diagnostic value of a “wide‐range” quantitative nested real‐time PCR assay for varicella zoster virus myelitis

Myelitis is one of the rarest neurological complications of varicella zoster virus (VZV) infection. In this study, the authors remodeled the "wide-range" quantitative nested real-time (QNRT) polymerase chain reaction (PCR) assay to quantitatively detect a small amount of VZV-DNA in cerebrospinal fluid (CSF). For use as a specific internal control "calibrator," an original mutation-VZV (MZ) plasmid was developed. The initial copy number of VZV-DNA in CSF specimens was measured by the amplification rate of the MZ-plasmid. For 17 consecutive CSF specimens collected from three elderly patients with VZV myelitis, the diagnostic value of the wide-range QNRT-PCR assay was evaluated and compared with other conventional PCR assays and enzyme immunoassay (EIA). The MZ-plasmid demonstrated statistically uniform amplifications (F=1.016) against a wide range (1-100,000) of copy numbers of mimic VZV-DNA. The wide-range QNRT-PCR assay quantitatively and rapidly (within 48 hr) detected 5,863, 3,052, 958, and 6,721 copies/ml of VZV-DNA in the CSF specimens collected from all patients in the acute phase. Additionally, there was a significant difference (*P=0.023) in the copy number of VZV-DNA between before and after acyclovir treatment. Other conventional single PCR assays all revealed negative results, but were nevertheless time-consuming (7 days). The IgG EIA-value for VZV was continually elevated throughout the clinical course of all patients. The MZ-plasmid was thus regarded as an appropriate "calibrator" in the wide-range QNRT-PCR assay. This assay is a novel, rapid, accurate, quantitative, and highly sensitive technique, and will contribute as a reliable and useful clinical examination for the rapid diagnosis of VZV infection to central nervous system.

Viral-bacterial associations in acute apical abscesses

Viral-bacterial and bacterial synergism have been suggested to contribute to the pathogenesis of several human diseases. This study sought to investigate the possible associations between 9 candidate endodontic bacterial pathogens and 9 human viruses in samples from acute apical abscesses. DNA extracts from purulent exudate aspirates of 33 cases of acute apical abscess were surveyed for the presence of 9 selected bacterial species using a 16S ribosomal RNA gene-based nested polymerase chain reaction (PCR) approach. Single or nested PCR assays were used for detection of the human papillomavirus (HPV) and herpesviruses types 1 to 8. Two-thirds of the abscess samples were positive for at least one of the target viruses. Specifically, the most frequently detected viruses were HHV-8 (54.5%); HPV (9%); and varicella zoster virus (VZV), Epstein-Barr virus (EBV), and HHV-6 (6%). Bacterial DNA was present in all cases and the most prevalent bacterial species were Treponema denticola (70%), Tannerella forsythia (67%), Porphyromonas endodontalis (67%), Dialister invisus (61%), and Dialister pneumosintes (57.5%). HHV-8 was positively associated with 7 of the target bacterial species and HPV with 4, but all these associations were weak. Several bacterial pairs showed a moderate positive association. Viral coinfection was found in 6 abscess cases, but no significant viral association could be determined. Findings demonstrated that bacterial and viral DNA occurred concomitantly in two-thirds of the samples from endodontic abscesses. Although this may suggest a role for viruses in the etiology of apical abscesses, the possibility also exists that the presence of viruses in abscess samples is merely a consequence of the bacterially induced disease process. Further studies are necessary to clarify the role of these viral-bacterial interactions, if any, in the pathogenesis of acute apical abscesses.

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 gene copy number aberrations in mantle cell lymphoma by a single quantitative multiplex PCR assay: clinicopathological relevance and prognosis value

The t(11;14)(q13;q32) is the hallmark of mantle cell lymphoma (MCL). Additional genetic alterations occur in the majority of cases. This study aimed to design a polymerase chain reaction (PCR) assay to determine the incidence and relevance of recurrent gene copy number aberrations in this disease. Forty-two MCL cases with frozen- or paraffin-embedded (FFPE) tissues were selected. Three different quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) assays were designed to simultaneously analyse eight genes (CDKN2A, RB1, ATM, CDK2, TP53, MYC, CDKN1B, MDM2), to analyse the 9p21 locus (CDKN2A/CDKN2B) and FFPE tissues. Gains of MYC, CDK2, CDKN1B, and MDM2 were observed in 10% of cases. Losses of RB1, CDKN2A, ATM or TP53 were observed in 38%, 31%, 24% and 10% of cases, respectively. Analysis of the 9p21 locus indicated that, in most cases, tumours displayed a complete inactivation of p14(ARF)/p15I(NK4B)/p16I(NK4A). CDKN2A and MYC aberrations were associated with a high MCL international prognostic index (MIPI). CDK2/MDM2 gains and CDKN2A/TP53 losses correlated with an unfavourable outcome. PCR experiments with frozen and FFPE-tissues indicated that our approach is valid in a routine diagnostic setting, providing a powerful tool that could be used for patient stratification in combination with MIPI in future clinical trials.

Application of an optimized 18-h method involving one step culturing and single primer-based PCR assay for detection of Salmonella spp. in foods

A polymerase chain reaction (PCR) using 20-mer oligonucleotide single primer (named primer 3) randomly designed on the basis of Salmonella Typhimurium gatD gene encoding galactitol-1-phosphate dehydrogenase could produce the specific DNA product of approximate 770-bp in all 38 Salmonella strains used. No 770-bp DNA band was amplified from any DNA samples of 20 non- Salmonella bacteria. The DNA band was detected by 1% agarose gel electrophoresis and ethidium bromide staining. The sensitivity of the RAPD–PCR assay for detection of pure genomic DNA from S. Typhimurium ATCC 13311 and Salmonella Enteritidis DMST 15676 was as few as 0.01 ng. When simple boiling in TE buffer for 5 min was used for extraction both Salmonella spp. DNA, the detection limits were at least 230 and 320 cells, respectively. By using the RAPD–PCR assay following at least 14 h pre-enrichment in nutrient broth (NB), as few as 1 CFU of S. Typhimurium ATCC 13311 or S. Enteritidis DMST 15676 per 25 g of autoclaved chicken meat was detected. When the optimized 18-h method involving pre-enrichment in NB and DNA extraction by boiling protocol followed by RAPD–PCR using primer 3 was evaluated in comparison with the conventional method on 195 possibly naturally-contaminated food samples, 36 samples were found positive by both methods. In addition, the results of the developed RAPD–PCR-based assay proved to be identical to those by the conventional method. The optimized 18-h method was simple, rapid and sensitive, achieved the same detection limit as the conventional method and produced a zero level of false-negative results.

Occult hepatitis B virus infection in Southern African blacks with hepatocellular carcinoma

To ascertain the prevalence of occult hepatitis B virus (HBV) infection in southern African blacks with hepatocellular carcinoma. Sera from 118 patients negative for HBV surface antigen but positive for HBV antibodies were studied. HBV-DNA was detected using a nested polymerase chain reaction (PCR) assay and confirmed by nucleotide sequencing of the surface and precore/core genes. Surface gene HBV-DNA was detected in a single PCR assay in 48.4% of the patients. Positive results increased to 57.7% after two PCR assays (not significant) and 75.7% after four assays (P < 0.001). No false positive results were obtained in these assays or in the 15 control samples for which PCR assays were performed four times. Significant differences in positivity rates were not observed between patients positive for HBV core antibody alone and those positive for core and surface antibodies. The sensitivity of the PCR amplification of the precore/core gene was significantly less than that of the surface open reading frame: the yield of positive results was 23.7% after one assay, 32.2% after two assays (not significant), and 52% after four assays (P < 0.001). Combining the results of the assays of the two genes increased the yield of positive results for the first assay (by 11.9%, P = 0.015), but not the second (6.1%) or fourth assays (4.6%). Occult HBV infection is present in the serum of the majority of hepatocellular carcinomas in southern African blacks whose serum is negative for hepatitis B surface antigen but positive for anti-HBV core antigen. The yield of positive results increases if more than one PCR assay is performed.

Preimplantation genetic diagnosis for Duchenne muscular dystrophy by multiple displacement amplification

To evaluate the use of multiple displacement amplification (MDA) in preimplantation genetic diagnosis (PGD) for female carriers with Duchenne muscular dystrophy (DMD). MDA was used to amplify a whole genome of single cells. Following the setup on single cells, the test was applied in two clinical cases of PGD. One mutant exon, six short tandem repeats (STR) markers within the dystrophin gene, and amelogenin were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products of single blastomeres. Center for reproductive medicine in First Affiliated Hospital, Sun Yat-sen University, China. Two female carriers with a duplication of exons 3-11 and a deletion of exons 47-50, respectively. The MDA of single cells and fluorescent PCR assays for PGD. The ability to analyze single blastomeres for DMD using MDA. The protocol setup previously allowed for the accurate diagnosis of each embryo. Two clinical cases resulted in a healthy girl, which was the first successful clinical application of MDA in PGD for DMD. We suggest that this protocol is reliable to increase the accuracy of the PGD for DMD.

Detection of Microsatellite Instability in Colorectal Cancer Using an Alternative Multiplex Assay of Quasi-Monomorphic Mononucleotide Markers

Colorectal malignancies demonstrating microsatellite instability (MSI) have a very heterogeneous histological appearance, better prognosis, and altered response to therapy. Consequently, identification of the MSI phenotype is both relevant and interesting as a screening and prognostic tool and as a potential predictive factor of chemotherapeutic response. Several groups have argued for the exclusive use of mononucleotide markers for MSI analysis. In this study, an alternative MSI typing multiplex system of mononucleotide microsatellite repeats was developed. This system obviates the need to compare allelic profiles between tumor and matching normal DNA, rendering MSI analysis amenable to high throughput. The quasi-monomorphic allelic distribution of five alternative mononucleotide markers was evaluated in genomic DNA. Only SEC63 and CAT25 were found to be quasi-monomorphic and were thus combined with BAT25 and BAT26 from the Bethesda panel. Consequently, 177 colorectal cancer samples previously analyzed by the Bethesda panel were tested for MSI using this alternative mononucleotide panel. In an attempt to resolve discordant cases, immunohistochemistry of MLH1, MSH2, and MSH6 was performed. The concordance between both panels reached 99.4% when microsatellite stability and MSI-L were grouped together. These new markers were subsequently multiplexed in a single polymerase chain reaction assay. The resulting mononucleotide fluorescent multiplex MSI assay has high accuracy, reliability, and throughput, thus reducing the time and cost involved in MSI testing.

Detection and Characterization of cry1Ac Transgene Construct in Bt Cotton: Multiple Polymerase Chain Reaction Approach

To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach can be adopted as a standard procedure for complete molecular characterization of Bt cotton. These assays will be of interest and use to importers, breeders, research laboratories, safety regulators, and food processors for detection of cry1Ac bearing GMOs.