Qualitative Polymerase Chain Reaction Methods Research Articles

Identification of Escherichia coli Sub Type Enterotoxigenic (ETEC) from Food Samples Using Pcr (Polymerase Chain Reaction) Technique

Escherichia coli is a normal flora of the digestive tract of humans and animals and is important in the digestion of food. This bacteria lives in feces, can cause health problems, such as diarrhea and vomiting. This research was conducted for consideration in the prevention of diarrhea, processing, marketing of food. This study aims to determine the presence of enterotoxigenic Escherichia coli sub-type (ETEC) from food samples. This research is an explorative descriptive study, as many as 5 samples from food, food samples taken are food consumed every day and some include traditional food in Mataram City. The samples were cultured on BHI-Broth media, followed by inoculation on Eosin Methylene Blue (EMB) medium and continued with the Gram stain test. Colonies that are metallic green in colour and show the characteristics of Escherichia coli bacteria on Gram staining are then carried out for purification of the bacteria on Nutrient Agar (NA) medium, if the classification of bacteria shows the characteristics of Escherichia coli bacteria on Gram staining then proceed with the Indole/sugar-test. The results showed that of the 5 food samples isolated, only 1 sample showed positive E.coli bacteria. Then proceed to the PCR (Polymerase Chain Reaction) stage using the qualitative PCR method to determine the presence of Enterotoxigenic Escherichia coli sub-type (ETEC) bacteria in food samples. , with a target gene of 237 bp.

Urinary circulating DNA and circulating antigen for diagnosis of schistosomiasis mansoni: a field study.

To evaluate three non-invasive assays for the diagnosis of schistosomiasis mansoni in an Egyptian village. Urine was collected for the detection of circulating cathodic antigen (CCA) and cell-free parasite DNA (cfpd) by Point-of-contact (POC)-cassette assay and PCR, respectively. These tests were compared to Kato-Katz (KK) faecal thick smear for detection of Schistosoma mansoni eggs. Disease prevalence by POC-CCA assay was 86%; by PCR it was 39% vs. 27% by KK. Compared to KK, the sensitivity of POC-CCA reached 100%, but its specificity was only 19.2% with 41% accuracy. Sensitivity of the PCR assay for cfpd was 55.56%, and specificity was 67.12% with 64% accuracy. A new end point was calculated for combined analysis of KK, POC-CCA assay and PCR. Sensitivity for the three tests was 52.94%, 90.2% and 76.47%; specificity was 100% for KK and PCR and 18.37% for POC-CCA. The accuracy calculated for the three tests at the end point was 76% for KK, 55% for POC-CCA assay and 88% for PCR. Conventional PCR assay for detection of cfpd provides a potential screening tool for intestinal schistosomiasis with reliable specificity, reasonable accuracy and affordable financial and technical cost.

Pooled Nucleic Acid Testing to Detect Antiretroviral Treatment Failure in HIV-Infected Patients in Mozambique.

In resource-limited settings, viral load monitoring of HIV-infected patients receiving antiretroviral therapy (ART) is not readily available because of high costs. Here, we compared the accuracy and costs of quantitative and qualitative pooled methods with standard viral load testing. Blood was collected prospectively from 461 patients receiving first-line ART in Mozambique who had not been evaluated previously with viral load testing. Screening for virologic failure of ART was performed quantitatively (ie, standard viral loads) and qualitatively [one and 2 rounds of polymerase chain reaction (PCR)]. Individual samples and minipools of 5 samples were then analyzed using both methods. The relative efficiency, accuracy, and costs of each method were calculated based on viral load thresholds for ART failure. Standard viral load testing of individual samples revealed a high rate of ART failure (19%-23%) across all virologic failure thresholds, and the majority of the patients (93%) with viral loads >1500 copies per milliliter had genotypic resistance to drugs in their ART regimen. Pooled quantitative screening and deconvolution testing had positive and negative predictive values exceeding 95% with cost savings of $11,250 compared with quantitative testing of each sample individually. Pooled qualitative screening and deconvolution testing had a higher cost savings of $30,147 for 1 PCR round and $25,535 for 2 PCR rounds compared with quantitative testing each sample individually. Both pooled qualitative PCR methods had positive and negative predictive values ≥90%, but the pooled 1-round PCR method had a sensitivity of 64%. Given the high rate of undiagnosed ART failure and drug resistance in this cohort, it is clear that virologic monitoring is urgently needed in this population. Here, we compared alternative methods of virologic monitoring with standard viral load testing of individual samples and found these methods to be cost saving and accurate. The test characteristics of each method will likely need to be considered for each local population before it is adopted.

Establishment and Application of Event-Specific Polymerase Chain Reaction Methods for Two Genetically Modified Soybean Events, A2704-12 and A5547-127

For implementation of the issued regulations and labeling policies for genetically modified organism (GMO) supervision, the polymerase chain reaction (PCR) method has been widely used due to its high specificity and sensitivity. In particular, use of the event-specific PCR method based on the flanking sequence of transgenes has become the primary trend. In this study, both qualitative and quantitative PCR methods were established on the basis of the 5' flanking sequence of transgenic soybean A2704-12 and the 3' flanking sequence of transgenic soybean A5547-127, respectively. In qualitative PCR assays, the limits of detection (LODs) were 10 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127. In quantitative real-time PCR assays, the LODs were 5 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127, and the limits of quantification (LOQs) were 10 copies for both. Low bias and acceptable SD and RSD values were also achieved in quantification of four blind samples using the developed real-time PCR assays. In addition, the developed PCR assays for the two transgenic soybean events were used for routine analysis of soybean samples imported to Shanghai in a 6 month period from October 2010 to March 2011. A total of 27 lots of soybean from the United States and Argentina were analyzed: 8 lots from the Unites States were found to have the GM soybean A2704-12 event, and the GM contents were <1.5% in all eight analyzed lots. On the contrary, no GM soybean A5547-127 content was found in any of the eight lots. These results demonstrated that the established event-specific qualitative and quantitative PCR methods could be used effectively in routine identification and quantification of GM soybeans A2704-12 and A5547-127 and their derived products.

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.

Effect of Prevention of the Mother to Child Transmission Program on the Prevalence of Postnatal HIV Infection in Benin City, Nigeria

The aim of this study was to determine the effect of prevention of the mother to child transmission (PMTCT) program in Benin City, Nigeria on the prevalence of postnatal infant HIV. Effects of the duration of PMTCT, place of birth and sex of the infants on the prevalence of postnatal HIV were also assessed. Dried blood spots were collected from 318 infants (6–8 weeks old) born to HIV-positive mothers and were screened for the presence of HIV using a qualitative polymerase chain reaction method. A questionnaire was used to obtain information about the duration of highly active antiretroviral therapy (HAART) and place of delivery. Male infants had a significantly ( p = 0.032) higher prevalence of postnatal HIV infection than females. Nonparticipation of mothers in the PMTCT program was a significant risk factor for acquiring postnatal HIV (odds ratio = 4.519; 95% confidence interval = 2.422, 8.429; p < 0.0001). The duration of HAART use significantly ( p = 0.010) affected the prevalence of postnatal HIV with an inverse relationship. The place of delivery had no effect on the prevalence of postnatal HIV. An overall prevalence of 16.98% of postnatal HIV was observed in this study. Male sex and no participation in the PMTCT program were significant risk factors for acquiring postnatal HIV, while a lower prevalence of postnatal HIV infection was associated with longer use of HAART in the PMTCT program. We recommend an early diagnosis of maternal HIV status and commencement of HAART.

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.

Event-specific analytical methods for biotech maize MIR 604 and DAS-59122-7

BACKGROUND: It is very important to develop analytical methods for genetically modified organism (GMO) labelling systems and living modified organism (LMO) management. The polymerase chain reaction (PCR) is the most efficient DNA-based analytical method for identifying and quantifying biotech crops. Qualitative PCR methods have been developed to detect the presence of biotech crops, while quantitative PCR methods have been developed to analyse the content of biotech crops. Analytical methods are now required for new biotech maize events, MIR 604 and DAS-59122-7. RESULTS: The event-specific primers and probes were developed for qualitative and quantitative analysis of biotech maize MIR 604 and DAS-59122-7 based on the 3′ flanking regions. As a reference molecule, single standard plasmid was developed. The specificity of the qualitative primers was confirmed by the single PCR product, with limits of detection (sensitivity) of 0.01 and 0.05% respectively. In-house validation of the quantitative methods was performed using six levels of mixing samples (0.1–10% w/w). As a result, the biases from the true value and the relative standard deviations were within the range of ± 30%. The limits of quantitation of the quantitative methods were all 0.1% for real time PCRs of MIR 604 and DAS-59122-7. CONCLUSION: In this study, event-specific analytical methods were developed and applied to the qualitative and quantitative analysis of biotech maize MIR 604 and DAS-59122-7. Copyright © 2009 Society of Chemical Industry

Qualitative Polymerase Chain Reaction Methods for Detecting Major Food Allergens (Peanut, Soybean, and Wheat) by Using Internal Transcribed Spacer Region

Allergen detection methods for peanut, soybean, and wheat were developed by designing PCR primer pairs for specific amplification of a fragment of the internal transcribed spacer (ITS) region reported for Arachis spp. for peanut, Glycine spp. for soybean, and Triticum and Aegilops spp. for wheat. The target species for detection included not only cultivated, but also wild and ancestor species, which were thought to be potentially allergenic. The ability of the resultant primer pairs to detect the target species was verified using genomic DNA extracted from A. hypogaea for peanut and G max for soybean; T. aestivum, T. turgidum, T. durum, T. aestivum-rye amphidiploid, T. monococcum, T. timopheevi, Ae. speltoides, and Ae. squarrosa for wheat. The LODs were 50-500 fg of target DNA, which were comparable to those of the most sensitive PCR methods previously reported. The results from the present work, as well as those from our previous work on buckwheat and kiwifruit, prove that the ITS region, for its high copy number and interspecific diversity, is particularly useful as the target of allergen detection methods.

Qualitative and quantitative PCR methods using species-specific primer for detection and identification of wood rot fungi

Species-specific oligonucleotide primers for detecting wood rot fungi, Gloeophyllum trabeum, Trametes versicolor, Coniophora puteana, and Serpula lacrymans, and the primer detecting a group of related fungi to G. sepiarium were developed. These primer sequences were picked up from the internal transcribed spacer region between small-subunit rDNA and large-subunit rDNA. The species selectivities of the developed primers were checked. Real-time polymerase chain reaction (PCR) was carried out using these highly specific primers to quantitatively detect at least of 0.01 ng genome DNA of the target species. This quantitative PCR was also used to differentiate the target species DNA from mixed species DNA. A PCR-based technique using the species-specific primers would be applicable to multiple-sample assay in diagnosis of wood decay and to investigation of environmental fungal populations.

A polymerase chain reaction directed to detect wheat glutenin: Implications for gluten-free labelling

Gluten enteropathy or celiac disease (CD) is treated by strict adherence to gluten-free diet for life. Trace amounts of wheat in food from farm to table manifests as a major risk to these individuals. A qualitative polymerase chain reaction method was developed by selectively amplifying a 135 bp fragment of the glutenin gene to detect wheat DNA in a plethora of raw and heat processed foods. The limit of detection was 21.5 pg of DNA. The absence of amplification in other cereals such as oat, rye, barley and maize renders this method exclusive for detection of wheat. The detection of wheat DNA in thermally processed foods by this method, despite extensive DNA fragmentation, evinces the suitability and applicability of the method for labeling gluten-free foods. This method complements the immunochemical methods toward addressing food safety in CD patients and wheat allergics.

Detection of Genetically Modified Organisms in Foreign-made Processed Foods Containing Corn and Potato

Investigations of the validity of labeling regarding genetically modified (GM) products were conducted using polymerase chain reaction (PCR) methods for foreign-made processed foods made from corn and potato purchased in the Tokyo area and in the USA. Several kinds of GM crops were detected in 12 of 32 samples of processed corn samples. More than two GM events for which safety reviews have been completed in Japan were simultaneously detected in 10 samples. GM events MON810 and Bt11 were most frequently detected in the samples by qualitative PCR methods. MON810 was detected in 11 of the 12 samples, and Bt11 was detected in 6 of the 12 samples. In addition, Roundup Ready soy was detected in one of the 12 samples. On the other hand, CBH351, for which the safety assessment was withdrawn in Japan, was not detected in any of the 12 samples. A trial quantitative analysis was performed on six of the GM maize qualitatively positive samples. The estimated amounts of GM maize in these samples ranged from 0.2 to 2.8%, except for one sample, which contained 24.1%. For this sample, the total amount found by event-specific quantitative analysis was 23.8%. Additionally, Roundup Ready soy was detected in one sample of 21 potato-processed foods, although GM potatoes were not detected in any sample.

Validation of a Tomato-Specific Gene, LAT52, Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Transgenic Tomatoes

Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR) detection methods of transgenic tomatoes, one tomato (Lycopersicon esculentum) species specific gene, LAT52, was selected and validated as suitable for using as an endogenous reference gene in transgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with 16 different tomato varieties, and identical amplified products or fluorescent signals were obtained with all of them. No amplified products and fluorescent signals were observed when DNA samples from 20 different plants such as soybean, maize, rapeseed, rice, and Arabidopsis thaliana were used as templates. These results demonstrated that the amplified LAT52 DNA sequence was specific for tomato. Furthermore, results of Southern blot showed that the LAT52 gene was a single-copy gene in the different tested tomato cultivars. In qualitative and quantitative PCR analysis, the detection sensitivities were 0.05 and 0.005 ng of tomato genomic DNA, respectively. In addition, two real-time assays employing this gene as an endogenous reference gene were established, one for the quantification of processed food samples derived from nontransgenic tomatoes that contained degraded target DNA and the other for the quantification of the junction region of CaMV35s promoter and the anti-sense ethylene-forming enzyme (EFE) gene in transgenic tomato Huafan No. 1 samples. All of these results indicated that the LAT52 gene could be successfully used as a tomato endogenous reference gene in practical qualitative and quantitative detection of transgenic tomatoes, even for some processed foods derived from transgenic and nontransgenic tomatoes.

Qualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes.

Qualitative and quantitative polymerase chain reaction (PCR) methods have been developed for the detection of genetically modified (GM) potatoes. The combination of specific primers for amplification of the promoter region of Cry3A gene, potato leafroll virus replicase gene, and potato virus Y coat protein gene allows to identify each line of NewLeaf, NewLeaf Y, and NewLeaf Plus GM potatoes. Multiplex PCR method was also established for the simple and rapid detection of the three lines of GM potato in a mixture sample. For further quantitative detection, the realtime PCR method has been developed. This method features the use of a standard plasmid as a reference molecule. Standard plasmid contains both a specific region of the transgene Cry3A and an endogenous UDP-glucose pyrophosphorylase gene of the potato. The test samples containing 0.5, 1, 3, and 5% GM potatoes were quantified by this method. At the 3.0% level of each line of GM potato, the relative standard deviations ranged from 6.0 to 19.6%. This result shows that the above PCR methods are applicable to detect GM potatoes quantitatively as well as qualitatively.

Assessment of screening methods for the identification of genetically modified potatoes in raw materials and finished products.

Qualitative polymerase chain reaction methods for the detection of genetically modified potatoes have been investigated that can be used for screening purposes and identification of insect-resistant and virus-resistant potatoes in food. The presence of the nos terminator from Agrobacterium tumefaciens and the antibiotic marker gene nptII (neomycin-phosphotransferase II) was demonstrated in three commercialized Bt-potato lines (Monsanto Co., St. Louis, MO, USA) and one noncommercial GM-potato product (high amylopectin starch, AVEBE, Veendam, The Netherlands) and allows for general screening in foods. For further identification, specific primers for the FMV promoter derived from the figwort mosaic virus, the CryIIIA gene (delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis), potato leafroll virus replicase gene, and the potato virus Y coat protein gene, were designed. The methods described were successfully applied to processed potato raw materials (dehydrated potato powders and flakes), starch samples, and finished products.

Development of a polymerase chain reaction assay for detection of three canola transgenes

AbstractMany countries are developing or implementing regulatory requirements to monitor for the presence of genetically modified (GM) materials in seeds, grain, and derived food products using DNA and protein‐based methods. There is no published report on the detection of different GM transgenes in canola, and this study is aimed at developing qualitative PCR methods for the three major GM transgenes commercially available in canola. Primer sequences were generated from Gen‐Bank and previously published information to develop a polymerase chain reaction (PCR) detection method for Roundup Ready (glyphosate tolerance, GT73 event), Liberty Link (glufosinate ammonium tolerance, HCN92 event), and BX (Bromoxynil tolerance, OXY235 event) canola varieties. On using PCR, two primer pairs for each of the GT73 and HCN92 and one primer pair for OXY235 amplified specific amplicons for the three GM transgenes. All three GM transgenes were detected simultaneously by multiplexing the five primer pairs in a single PCR reaction. Multiplexing of the five primer pairs for DNA prepared at 1% (one GM seed in 99 non‐GM seeds) and 0.5% (one GM seed in 199 non‐GM seeds) levels generated the expected DNA fragments for GT73, HCN92, and OXY235. This information will lay the groundwork for the development of a quantitative PCR assay for canola transgenic events.

The validation of methods based on polymerase chain reaction for the detection of genetically modified organisms in food

Validated analytical methods are necessary in order to show compliance with labelling concerning conventionally produced food or food containing genetically modified organisms (GMOs). Methods to be used in arbitration should be collaboratively tested according to standardisation protocols. According to the Council Regulation of 1998, one of the marker components being the criterion for labelling of products from soy beans and maize is the genetically altered DNA. A qualitative polymerase chain reaction method which has recently been validated in a collaborative trial can be used for initial screening of food products in order to detect GMOs. Furthermore, this screening method has to be validated for processed food. In addition, there is a strong need for quantitative methods with a view to a threshold value for labelling and to distinguish between the source of GMOs in food products (ingredients or additives).

Utility of cytomegalovirus viral load in renal transplant patients in Argentina.

Cytomegalovirus (CMV) is the most prevalent viral disease in solid organ transplantation. Detection of CMV DNA in peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR) frequently occurs in renal allograft recipients, yielding false positive results in seropositive patients free of CMV disease. We evaluated the clinical utility of a quantitative PCR-enzyme-linked immunosorbent assay (ELISA) for identifying patients with CMV disease. Three hundred and fifty samples from 65 consecutive renal transplant recipients were studied. DNA was extracted from PBMC weekly up to the day of discharge and after any further admission. Samples were tested by a qualitative PCR method, and all positive samples were further studied by a quantitative PCR-ELISA method. The quantitative PCR-ELISA method used an internal standard (IS) that contained the primer sequences used in the qualitative CMV PCR. Detection and quantification was performed in 96-well plates coated with IS or CMV specific probes. Forty-one of 65 patients (63.1%) showed positive results by the qualitative PCR, but only 8 of these patients were diagnosed with CMV disease. Positive samples were re-analyzed by the quantitative assay. The 8 patients with CMV disease had a mean CMV viral load of 1,438+/-687 viral copies (VC)/10(6) PBMC, and the 33 without CMV disease had a mean value of 219.6+/-117.2 VC/10(6) PBMC (P<0.01). None of the 33 patients without CMV disease had viral loads higher than 500 VC/10(6) PBMC. Using 500 VC/10(6) PBMC as a cut-off value for CMV disease, the quantitative PCR showed a sensitivity and specificity of 100% compare to clinical diagnosis. CMV viral load may be useful in the diagnosis of CMV disease in renal transplant patients.