Quantitative Polymerase Chain Reaction Detection Research Articles

Human papillomavirus in oropharyngeal squamous cell carcinoma: Assessing virus presence in normal tissue and activity in cervical metastasis

Human papillomavirus (HPV) has been established as an etiologic and prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). HPV oncogenesis involves expression of E6/E7 oncoproteins, with downstream p53 degradation and pRb inhibition. Although much research has focused on HPV's oncogenic behavior in primary OPSCC, minimal information exists about HPV in adjacent normal and metastatic tissue. Retrospective cohort study Patient-matched tumor, normal, and metastatic tissue was gathered from 42 OPSCC patients and tested with real-time quantitative polymerase chain reaction (RT-qPCR), in situ hybridization (ISH), and immunohistochemistry (IHC). RT-qPCR was performed using total RNA from fresh-frozen tissues and primers for HPV16 E6, E7, and p16 transcripts. HPV ISH was performed to detect the presence of HPV DNA and IHC to detect p16 protein. Primary tumor, adjacent normal tissue, and tumor metastasis from 17 OPSCC patients were analyzed. When comparing the presence of HPV16 DNA in tumor, metastatic, and normal tissue by ISH, perfect correlation is found at all subsites (P < .0001). However, active infections determined by HPV16 E6 and E7 expression using quantitative polymerase chain reaction or p16 detection by IHC, were present only in primary and metastatic tissue (P = .0012, E6; P = .02, E7). No such correlation was found in normal tissue when compared to primary or metastatic tissue. There is a clear pattern of active HPV expression that correlates to disease course. In HPV-positive patients, all sites including primary, metastatic, and normal tissues are DNA positive. Transcriptionally active infections were detected in primary and metastatic tissues, whereas normal tissues appear to have latent infections.

Event-specific qualitative and quantitative polymerase chain reaction methods for detection of insect-resistant genetically modified Chinese cabbage based on the 3′-junction of the insertion site

Transgenic Chinese cabbage 416-3 was developed in Korea by a transformation event involving modified insectresistant gene (cry1Ac1). To monitor unintended release of genetically modified (GM) Chinese cabbage in the future, as well as to meet GM-labeling requirements, the development of a reliable method for detection of GM cabbage is requisite. To develop qualitative and quantitative polymerase chain reaction methods for the insect-resistant GM Chinese cabbage, a cytosolic glutathione reductase (BcgGR1) gene was used as the endogenous reference gene. Primer pairs CGR-1/-2, amplifying the Chinese cabbage endogenous gene, yielded an expected amplicon of 121 bp, whereas no amplified product was observed when DNA samples from seven non-cabbage plants were used as templates. The event-specific primer pairs amplifying the junction site between the endogenous genome sequence and the transferred DNA of GM event 416-3, produced amplicons of desired size by qualitative polymerase chain reaction (PCR) assay. An eventspecific quantitative PCR detection method was established using a TaqMan probe and a standard plasmid as a reference molecule, which contained both endogenous and event-specific sequences. For the validation of this method, three different compositions of w/w mixed samples (containing transgenic DNA at 5, 1, and 0.5% of total DNA in the control samples) were quantified. The precision, expressed as standard deviation (SD) and relative standard deviations (RSD), deviated by 0.03–0.26% and 4.75–8.06%, respectively. These results clearly demonstrate that the PCR methods developed herein can be used for event-specific qualitative and quantitative testings of insect-resistant GM Chinese cabbage.

Real‐time quantitative polymerase chain reaction detection of haemoplasmas in healthy and unhealthy dogs from Central Macedonia, Greece

The aims of this study were to determine the prevalence of canine haemoplasmas, Mycoplasma haemocanis and "Candidatus Mycoplasma haematoparvum" infection in Central Macedonia, Greece, and to evaluate any associations between canine haemoplasma infection and clinical presentation, selected laboratory data or the presence of ticks. Genomic DNA was purified from excess blood (n=151) submitted for haematological examination. Purified DNA was subjected to species-specific quantitative polymerase chain reaction assays duplexed with a canine DNA control quantitative polymerase chain reaction. Clinical records were retrospectively examined and selected clinical parameters were compared to haemoplasma infection status. Nine samples were excluded due to inadequate canine DNA polymerase chain reaction results. Of the remaining 142 samples: eight (5·6%) were positive for M. haemocanis alone, six (4·2%) were positive for "Ca. M. haematoparvum" alone and one (0·7%) was dual positive. No association was found between haemoplasma status and age, sex, breed, health status, presence of anaemia, selected biochemistry parameters, presence of ectoparasites, routine ectoparasiticide treatment or the presence of selected tick-borne diseases.

Rapid, specific and quantitative polymerase chain reaction (PCR) detection of pathogenic protozoa Entamoeba histolytica for drinking water supply

In this study, a quantitative species-specific polymerase chain reaction (PCR) method to rapidly detect E. histolytica in water is developed. First, the specificity of E. histolytica PCR detection was verified by using species-specific primers of 16S-like rRNA genes to clearly differentiate it from the closely related amoebae species E. dispar and E. moshkovskii. The sensitivity of this method was subsequently determined using purified E. histolytica genomic DNA and culture cells as PCR reaction templates. Results indicated that conventional PCR visualized on 1% agarose gel was able to detect as low as 0.02 pg genomic DNA and 5 cells, while real-time PCR could detect 0.01 pg genomic DNA and 2 cells of E. histolytica. The protocols for E. histolytica PCR detection in real water samples were then optimized by spiking E. histolytica cells into tap water and reservoir raw water samples. A two-round centrifugation treatment to concentrate amoeba cells directly as a PCR template was the most effective way to detect E. histolytica in spiked tap water samples, while DNA extraction after concentrating amoeba cells was required for spiked reservoir raw water samples. The detection limit of 50 E. histolytica cells in 100 ml tap water was achieved in 2 h from sample collection to real-time PCR data readout. With these established protocols, 78 tap water samples, 11 reservoir raw water samples and 4 feed water samples from Singapore water supply systems were analyzed by both conventional PCR and real-time PCR methods. No E. histolytica cell was detected in tested samples.

Establishment and evaluation of event-specific qualitative and quantitative PCR method for genetically modified soybean DP-356043-5

With the increasing development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. The polymerase chain reaction (PCR) technique has been the mainstay for GMO detection, especially for event-specific qualitative and quantitative PCR detection methods, which have become the internationally agreed state-of-art. This paper describes the character and event-specific quantitative detection method of DP-356043-5 (356043) soybean. In this research, the flanking regions were characterized by inverse PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right and left flanking sequences. In the qualitative PCR assay, PCR systems were established with the species-specific and event-specific primers, respectively. And event-specific primers were established on both right and left flanking sequences; the limit of detection (LOD) was both 0.05% (approximates to 42 haploid genome copies). In the quantitative TaqMan real-time PCR assay, we obtained standard curves with good linearity and relatively high efficiency of PCR. All the results indicated that the established event-specific qualitative and quantitative PCR systems for 356043 soybean in this study were reliable and suitable for 356043 soybean detection in mixed samples. Besides, based on the flanking sequence information we obtained, not only the qualitative and quantitative PCR system for detecting 356043 soybean can be established, but also some other novel event-specific detection methods using gene microarray, biosensor, etc., with target sequence on them can also be developed, which have a good value for detecting 356043 soybean.

Overexpression of the Parkinson Disease Protein DJ-1 and its Regulator PTEN in Gestational Trophoblastic Disease

DJ-1 is found to be important in human neurodegenerative diseases and cancers by regulating oxidative damage and apoptosis. DJ-1 is also a regulator of PTEN, a frequently mutated tumor suppressor gene in cancers. In this study, we investigated the expression of DJ-1 and PTEN in normal placentas and gestational trophoblastic disease in relation to apoptotic indices and p53 status. A total of 95 trophoblastic samples were retrieved for immunohistochemical study whereas 79 trophoblastic samples, 3 normal trophoblastic and 2 choriocarcinoma cell lines were collected for quantitative real time reverse transcription polymerase chain reaction detection. There was a significant correlation between DJ-1 and PTEN immunostaining indices in the trophoblastic samples (P=0.013). Significantly higher DJ-1 and PTEN immunoreactivity indices were found in the complete mole (P<0.01) and choricarcinoma (P<0.01) compared with the first trimester placenta. Quantitative real time reverse transcription polymerase chain reaction also detected significantly higher messenger ribonucleic acid expressions of DJ-1 and PTEN in hydatidiform moles (P<0.05) and choriocarcinomas (P<0.05) compared with the first trimester placentas. A significant negative correlation was found between DJ-1 and the apoptosis resistant gene Bcl-2 (P=0.031), whereas a positive correlation was shown between PTEN and wild-type p53 (P=0.019). Significant correlations between PTEN and embryonic stem cell transcription factors, Stat3 and Nanog, were also displayed (P=0.001, 0.015). Our findings showed, for the first time, overexpression of DJ-1 at both transcriptional and protein levels in gestational trophoblastic disease. Overexpressed DJ-1 may play a role in regulating apoptotic activities of trophoblasts in relation to PTEN and p53.

Bladder tumor targeted expression of adenovirus mediated SEA gene regulated by hTERT and PSA promoters

Objective SEA gene construct carrying the selective adenovirus, in vitro experimental observation SEA gene expression and to stimulate anti-tumor function of lymphocytes. Methods Building a dual boot by telomerase, and HIF-carrying SEA selective adenovirus by real-time quantitative polymerase chain reaction (PCR) detection of SEA in the nucleic acid expression in tumor cells, immune fluorescence positioned SEA expressed in tumor cells, Western blotting determination of protein expression, observed under a microscope, dynamic co-culture of lymphocytes and tumor cells, enzyme linked immu-nosorbent assay (ELISA) detection of interleukin (IL)-4 secretion. Results SEA levels of success in the nucleic acid and protein expression, cell ELISA and flow cytometry proved to be CD3+ T lymphocytes significantly stimulated proliferation, lymphocytes and tumor cells co-cultured tumor cells 12, 24, 48 hours the experimental group significantly lower than the control group, ELISA detection of IL-4 secretion experi-mentsgroup were higher than that (t = 3.585 P < 0.05 ). Conclusion The choice of carrying SEA gene adenovirus gene was successfully constructed and expressed a preliminary verification of the tumor cells in vitro. Key words: Bladder tumor; Oncolytic adenovirus; Superantigen SEA

Development and in‐house validation of the event‐specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985

To implement genetically modified organism (GMO) labeling regulations, an event-specific analysis method based on the junction sequence between exogenous integration and host genomic DNA has become the preferential approach for GMO identification and quantification. In this study, specific primers and TaqMan probes based on the revealed 5'-end junction sequence of GM cotton MON15985 were designed, and qualitative and quantitative polymerase chain reaction (PCR) assays were established employing the designed primers and probes. In the qualitative PCR assay, the limit of detection (LOD) was 0.5 g kg(-1) in 100 ng total cotton genomic DNA, corresponding to about 17 copies of haploid cotton genomic DNA, and the LOD and limit of quantification (LOQ) for quantitative PCR assay were 10 and 17 copies of haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR assays were validated in-house by five different researchers. Also, five practical samples with known GM contents were quantified using the developed PCR assay in in-house validation, and the bias between the true and quantification values ranged from 2.06% to 12.59%. This study shows that the developed qualitative and quantitative PCR methods are applicable for the identification and quantification of GM cotton MON15985 and its derivates.

Characterization and event‐specific quantitative detection of DAS‐59122‐7 maize insert with the application of plasmidic reference material

AbstractBACKGROUND: With the development of genetically modified organisms (GMOs), event‐specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed standard.RESULTS: The flanking regions of DAS‐59122‐7 maize were characterized by inverse PCR (I‐PCR). In the qualitative PCR assay, a duplex PCR was established with the event‐specific and taxon‐specific primers, and the limit of detection (LOD) was 1 g kg−1 (approximates to 38 haploid genome copies). In the quantitative TaqMan® real‐time PCR assay, a plasmidic reference material was constructed by recombinant PCR and standard curves were set up. By using the plasmidic reference material, we obtained standard curves with good linearity and relatively high efficiency. The results indicated the usability of the plasmid as standard material.CONCLUSION: From above results, we believe that the developed event‐specific qualitative and quantitative PCR systems for DAS‐59122‐7 maize in this study are acceptable and suitable for DAS‐59122‐7 maize detection. Copyright © 2008 Society of Chemical Industry

Microchimerism decades after transfusion among combat‐injured US veterans from the Vietnam, Korean, and World War II conflicts

Blood transfusion after traumatic injury can result in microchimerism (MC) of donor white cells (WBCs) in the recipient as late as 2 to 3 years postinjury, the longest prospective follow-up to date. The purpose of this study was to determine how long transfusion-associated MC lasts after traumatic injury. A group of US combat veterans who received transfusions who responded to a recruitment notice was retrospectively evaluated. Their blood was sampled, and MC was assessed by quantitative allele-specific polymerase chain reaction detection of differences at the HLA-DR locus or a panel of insertion-deletion polymorphism loci. Results of veterans were compared to those from an age- and gender-matched blood donor control group, from whom WBCs were retrieved from leukoreduction filters. Among 163 combat veterans who received transfusion and 150 control subjects who did not receive transfusions, 16 (9.8%) of the veterans and 1 (0.7%) control subject had evidence of MC (relative risk, 14.7; 95% confidence interval, 2.0-110). The veterans with MC included 3 who served in WWII (7% of subjects from that conflict), 5 in Korea (18%), and 6 in Vietnam (7%). Transfusion for combat-related injury can result in MC that lasts for 60 years, suggesting that it may involve permanent engraftment. MC is rare among male blood donors who did not receive transfusions, who are probably representative of individuals who have not had postnatal allogeneic exposures.

Quantification of enterococci and bifidobacteria in Georgia estuaries using conventional and molecular methods

Fecal pollution is a serious threat to the estuarine environment along the Georgia coast. Culture-dependant and molecular methodologies were utilized to compare and evaluate the abundance of fecal indicator bacteria in four Georgia estuaries (Darien River, Frederica River, Gulley Hole Creek, and St. Marys River). The functionality of enterococci and bifidobacteria as indicator organisms in marine environments was assessed, as well as Bifidobacterium adolescentis densities. At each study site, enterococci were enumerated as colony forming units (CFU) on mEI agar. For quantitative polymerase chain reaction (qPCR), genus- and species-specific primer sets were used to quantify bifidobacteria and B. adolescentis as 16S rRNA gene copies and enterococci as tuf gene copies. A high correlation ( r = 0.925) was observed between CFU and qPCR enumeration of enterococci. Enterococci densities in the estuarine rivers ranged from 3–449 CFU/100 ml on mEI plates and 4.58–5.39 Log 10 gene copies/100 ml by qPCR. Bifidobacteria densities ranged from 3.62–4.14 Log 10 gene copies/100 ml and suggested the Frederica River as least affected by fecal bacteria and the Darien River as most affected by fecal pollution. A correlation of 0.46 was observed among qPCR densities of enterococci and bifidobacteria at all sample sites. Quantitative polymerase chain reaction detection of B. adolescentis was a rapid (i.e., less than 2 h) indicator of presumptive human fecal pollution and suggested that Gulley Hole Creek, the Darien River, and the St. Marys River were affected by fecal bacteria derived from a human source. Gulley Hole Creek and the Darien River had the highest levels of fecal pollution detected in the studied estuaries. Molecular quantification of bifidobacteria may be a more accurate method of determining immediate health risks associated with fecal pollution in estuarine water than traditional and contemporary assessments of enterococci.

Event-Specific Qualitative and Quantitative PCR Detection Methods for Transgenic Rapeseed Hybrids MS1×RF1 and MS1×RF2

Except for the events RT73, MS8, RF3, and T45, event-specific detection methods for most commercialized genetically modified (GM) rapeseed varieties have not been established, and as a result, the enforcement of genetically modified organism labeling policies has been hindered. The genetically modified rapeseeds, MS1xRF1 and MS1xRF2, are 2 of 11 approved GM-rapeseed varieties for commercialization. In this study, the right border junction fragments between the gene construct and the rapeseed genome of events RF1, RF2, and MS1 were isolated using the commercially available GenomeWalker technology. Homology analysis indicated that the gene construct of RF1 integrated upstream of the nuclease gene, and that of the RF2 and MS1 inserted into the exon region of a gene encoding for an unknown protein. The event-specific primer pairs and corresponding probes were designed on the basis of the revealed right border junction fragments. Then, we successfully developed the identification and quantification methods for the gene-stacked hybrids MS1xRF1 and MS1xRF2 using those primers and probes. The relative limit of detection in the qualitative polymerase chain reaction (PCR) was 0.013% for the RF2 and MS1 assays using 100 ng of rapeseed DNA per reaction and 0.13% for the RF1 assay. The absolute limit of detection in the quantitative PCR was approximately one to two initial copies for each of the three event-specific assays. The evaluation of the real-time PCR assays revealed that the qualitative and quantitative methods developed by focusing on the gene-stacked hybrids MS1xRF1 and MS1xRF2 were highly specific, sensitive, and suitable for samples with a low quantity of DNA.

Event-specific qualitative and quantitative PCR detection of roundup ready event GT73 based on the 3′-integration junction

With the development of genetically modified organisms, labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed state-of-art. This paper describes an event-specific PCR method for qualitative and quantitative of Roundup Ready canola event GT73. The 3'-integration junction was characterized by two methods: inverse-PCR and thermal asymmetric interlaced-PCR. In the conventional qualitative PCR assay, the event-specific primers designed were confirmed to be specific and the limit of detection (LOD) was 0.05% (approximates to ten haploid genome copies). In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were five and ten haploid genome copies, respectively. In addition, for further quantitative detection, a reference molecule which contained the canola endogenous gene and event-specific sequence was constructed and standard curves were set up. The goodness of the linearity and high efficiency of the PCR reaction indicated the usability of the plasmid and the established PCR system. Moreover, mixed samples with different GT73 content (6, 3, 1 and 0.5%) were quantified using the established real-time PCR system to evaluate the trueness and precision of the system. The trueness expressed as bias varied from 2.00 to 18.00%. The precision expressed as variation coefficient were different from 6.40 to 32.95%. From above results, we believed that the established event-specific qualitative and quantitative PCR systems for GT73 in this study were acceptable and suitable for genetic modified canola detection.

Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease implicated in amphibian declines on 5 continents. Polymerase chain reaction (PCR) primer sets exist with which amphibians can be tested for this disease, and advances in sampling techniques allow non-invasive testing of animals. We developed filtering and PCR based quantitative methods by modifying existing PCR assays to detect Bd DNA in water and sediments, without the need for testing amphibians; we tested the methods at 4 field sites. The SYBR based assay using Boyle primers (SYBR/Boyle assay) and the Taqman based assay using Wood primers performed similarly with samples generated in the laboratory (Bd spiked filters), but the SYBR/Boyle assay detected Bd DNA in more field samples. We detected Bd DNA in water from 3 of 4 sites tested, including one pond historically negative for chytridiomycosis. Zoospore equivalents in sampled water ranged from 19 to 454 1(-1) (nominal detection limit is 10 DNA copies, or about 0.06 zoospore). We did not detect DNA of Bd from sediments collected at any sites. Our filtering and amplification methods provide a new tool to investigate critical aspects of Bd in the environment.

Event-Specific Qualitative and Quantitative Polymerase Chain Reaction Analysis for Genetically Modified Canola T45

Polymerase chain reaction (PCR) methods have been the main technical support for the detection of genetically modified organisms (GMOs). To date, GMO-specific PCR detection strategies have been developed basically at four different levels, such as screening-, gene-, construct-, and event-specific detection methods. Event-specific PCR detection method is the primary trend in GMO detection because of its high specificity based on the flanking sequence of exogenous integrant. GM canola, event T45, with tolerance to glufosinate ammonium is one of the commercial genetically modified (GM) canola events approved in China. In this study, the 5'-integration junction sequence between host plant DNA and the integrated gene construct of T45 canola was cloned and revealed by means of TAIL-PCR. Specific PCR primers and TaqMan probes were designed based upon the revealed sequence, and qualitative and quantitative TaqMan real-time PCR detection assays employing these primers and probe were developed. In qualitative PCR, the limit of detection (LOD) was 0.1% for T45 canola in 100 ng of genomic DNA. The quantitative PCR assay showed limits of detection and quantification (LOD and LOQ) of 5 and 50 haploid genome copies, respectively. In addition, three mixed canola samples with known GM contents were detected employing the developed real-time PCR assay, and expected results were obtained. These results indicated that the developed event-specific PCR methods can be used for identification and quantification of T45 canola and its derivates.

Leukoreduction of blood transfusions does not diminish transfusion‐associated microchimerism in trauma patients

Transfusion of trauma patients can result in long-term survival of donor white blood cells (WBCs) or "transfusion-associated microchimerism" (TA-MC). The aim was to determine whether leukoreduction of blood transfusions, advocated to reduce the immunomodulatory effect of transfusion, decreases the likelihood of developing TA-MC. A subgroup of trauma patients from a randomized trial was examined, evaluating the risk of infection following leukoreduced versus nonleukoreduced blood transfusion. Patients' blood was sampled at least 1 month after hospital discharge, and TA-MC was assessed with quantitative allele-specific polymerase chain reaction detection of differences at the HLA-DR locus or a panel of insertion-deletion polymorphism loci distributed throughout the chromosomal complement. At the time of blood sampling, a scripted interview was used to ascertain symptoms suggestive of chronic graft-versus host disease (cGVHD). For 67 patients evaluated, the mean age was 43 +/- 17 years and mean Injury Severity Score was 24 +/- 12. Median time from injury to blood sampling for TA-MC was 240 (interquartile range, 116-360) days. Nine of 32 patients (28%) in the nonleukoreduced transfusion group developed TA-MC compared to 13 of 35 patients (37%) in the leukoreduced group (p = 0.43). Subjects with TA-MC were no more likely than subjects without TA-MC to have at least one symptom suggestive of cGVHD (64% vs. 76%, respectively). TA-MC seems to be a prevalent condition among injured patients at the second of two regional trauma centers evaluated, suggesting that it is a common phenomenon after transfusion in the setting of injury. Although leukoreduction removes greater than 99.9 percent of donor WBCs, it fails to prevent or even substantially reduce the likelihood of developing TA-MC. TA-MC does not appear to be strongly associated with symptoms suggestive of cGVHD several months after transfusion.

Herpes simplex encephalitis: Adolescents and adults

Herpes simplex encephalitis (HSE) remains one of the most devastating infections of the central nervous system despite available antiviral therapy. Children and adolescents account for approximately one third of all cases of HSE. Clinical diagnosis is suggested in the encephalopathic, febrile patient with focal neurologic signs. However, these clinical findings are not pathognomonic because numerous other diseases in the central nervous system can mimic HSE. Neurodiagnostic evaluation can provide support for the diagnosis by the demonstration of temporal lobe edema/hemorrhage by magnetic resonance image scan and spike and slow-wave activity on electroencephalogram. In the current era, the diagnostic gold standard is the detection of herpes simplex virus (HSV) DNA in the cerebrospinal fluid by polymerase chain reaction (PCR). Although PCR is an excellent test and preferable to brain biopsy, false negatives can occur early after disease onset. Acyclovir is the treatment of choice and is administered at 10 mg/kg every 8 h for 21 days. Even with early administration of therapy after the disease onset, nearly two thirds of survivors have significant residual neurologic deficits. Current investigative efforts are assessing the prognostic value of quantitative PCR detection of viral DNA at the onset of therapy as well as at the completion of therapy and the contribution of prolonged antiviral therapy to improved neurologic outcome.

A cotton‐specific gene, stearoyl‐ACP desaturase, used as a reference for qualitative and real‐time quantitative polymerase chain reaction detection of genetically modified organisms

AbstractBiotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)‐based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl‐ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real‐time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties. Copyright © 2006 Society of Chemical Industry

Event Specific Qualitative and Quantitative Polymerase Chain Reaction Detection of Genetically Modified MON863 Maize Based on the 5‘-Transgene Integration Sequence

Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.

Novel Reference Gene, High-mobility-group protein I/Y, Used in Qualitative and Real-Time Quantitative Polymerase Chain Reaction Detection of Transgenic Rapeseed Cultivars

With the development of transgenic crops, regulations to label the genetically modified organisms (GMOs) and their derived products have been issued in many countries. Polymerase chain reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods are generally needed to amplify the transgene and compare the amplified results with that of a corresponding reference gene to get the reliable results. Specific primers were developed for the rapeseed (Brassica napus), high-mobility-group protein I/Y(HMG-I/Y) single-copy gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 15 different rapeseed varieties, and identical amplified products were obtained with all of them. No amplification was observed when templates were the DNA samples from the other species of Brassica genus or other species, such as broccoli, stem mustard, cauliflower, Chinese cabbage, cabbage, sprouts, Arabidopsis thaliana, carrot, tobacco, soybean, mung bean, tomato, pepper, eggplant, plum, wheat, maize, barley, rice, lupine, and sunflower. This system was specific for rapeseed. Limits of detection and quantitation in qualitative and quantitative PCR systems were about 13 pg DNA (about 10 haploid genomes) and about 1.3 pg DNA (about 1 haploid genome), respectively. To further test the feasibility of this HMG-I/Y gene as an endogenous reference gene, samples containing transgenic rapeseed GT73 with the inserted glyphosate oxidoreductase (GOX) gene were quantitated. These demonstrated that the endogenous PCR detection systems were applicable to the qualitative and quantitative detection of transgenic rapeseed.