Direct Polymerase Chain Reaction Method Research Articles

Pupal Exuviae of Culex Pipiens L. (Diptera: Culicidae) Can be Utilised as a Non-Invasive Method of Biotype Differentiation

BackgroundCulex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay.ResultsGenomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55–80%.ConclusionsThis study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

Development of rapid on-site detection of Salmonella Enteritidis, S. Typhimurium, and S. Thompson in food samples using an ultrafast PCR system

The conventional Salmonella serotyping methods are labor intensive and time-consuming; therefore, rapid identification method is needed to detect outbreaks early and prevent their spread. In this study, we developed for the first time a detection method for the rapid and accurate on-site serotyping of Salmonella enterica subsp. enterica serovar Enteritidis, Thompson, and Typhimurium, all of which have epidemiological importance worldwide. This method, which is based on specific marker genes, was constructed with portable equipment and a simple DNA extraction step. For the specific detection of the three serovars, genetic markers obtained via a bioinformatics analysis were selected and showed high specificity for 97 strains. The ultrafast polymerase chain reaction (PCR) showed high linearity (≥0.997) in both pure culture and a spiked food sample, with a detection specificity of 102 colony forming unit (CFU)/ml; these results are consistent with real-time PCR. Moreover, ultrafast PCR was further improved for developing direct ultrafast PCR method for field detection, the entire procedure of which could be completed within 25 min. The test results of an artificially contaminated food sample showed that the three serovars could be detected using the direct ultrafast PCR with a very short (3 h) enrichment step when inoculated at 1 CFU/g. This method exhibited various advantages, such as its ultrafast and labor-saving characteristics, which can be applied to the diagnosis of Salmonella serovar in the field.

Development of a Simple Direct and Hot-Start PCR Using Escherichia coli-Expressing Taq DNA Polymerase.

Taq DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing Taq DNA polymerase for routine use in laboratories. We developed a method using Escherichia coli (E. coli) that expresses thermostable Taq DNA polymerase directly in the PCR without purification. The Taq gene was transformed into E. coli and expressed. After overnight incubation and washing, E. coli-expressing Taq DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.

SG-APSIC1123: Diagnostic and infection prevention and control (IPC) performance of rapid polymerase chain reaction (PCR) compared to conventional culture PCR methods for detecting carbapenemase-producing organisms (CPOs)

Objectives: In this study, we compared the performance of a rapid polymerase chain reaction (PCR) method in detecting carbapenemase-producing organisms (CPOs) and its impact on infection prevention and control (IPC) measures compared with a culture PCR method. Methods: All patients requiring CPO screening were included. Rectal swabs were collected with double rayon swabs (Copan 139C). They were simultaneously analyzed for the presence of CPOs using rapid PCR assay (Xpert Carba-R assay, Cepheid, Sunnyvale, CA) and a culture–PCR method (ChromID CARBA-SMART, bioMerieux, Marcy-l’Etoile, France). For CARBA-SMART, only colored colonies (ie, Enterobacterales) were evaluated for CPOs according to the prevailing institutional protocol. We tracked time to CPO detection. Using CPO positivity from either the rapid PCR or the culture PCR method as the gold standard, we calculated the sensitivity and specificity of both tests. We calculated the number of epidemiologically linked contacts generated when the first test results were known. We prospectively followed the ward census to identify the putative additional number of contacts generated by the later known result. Contacts were patients who shared the same ward (with overlapping time) as the CPO patients. Results: Between April 2019 and June 2020, culture PCR method detected CPOs in 316 (1.3%) of 24,514 samples (blaOXA48, N = 211; blaNDM, N = 51; blaIMI, N = 21; blaIMP, N = 10; blaKPC, N = 9; mixed genotypes, N = 14). The rapid PCR test detected CPOs in 605(2.5%) of 24,514 samples (blaOXA48, N = 266; blaNDM, N = 161; blaIMP, N = 99; blaVIM, N = 29; blaKPC, N = 15; mixed genotypes, N = 35). The sensitivity of direct PCR and culture PCR methods were 94.2% (95% CI, 92.1%–95.8%) and 43.5% (95% CI, 39.6%–47.4%), respectively. Both tests had 100% specificity. The median times to detection for the rapid PCR and culture PCR methods were 3–4 hours and 4 days, respectively. Compared with rapid PCR, the culture PCR method generated additional 7,415 contacts when it also tested positive for CPOs and an additional 23,135 contacts when it tested negative for CPOs. Conclusions: In our study, the rapid PCR test was more sensitive, identified CPO faster, and generated fewer epidemiologically linked contacts than the culture PCR method.

Improved direct lysis PCR amplification method of microalgal culture for sequencing and species identification

Molecular approach plays important role in species identification for microalgae which involves sequencing of specific DNA barcode present in the genome. This approach involved preparation of template DNA for polymerase chain reaction (PCR) which is time consuming and requires large amounts of algal cells. Microalgal direct PCR have been used frequently for species identification, which simplified the DNA isolation procedure. However, the recent attempts to amplify the rbcL gene of microalga using the previously reported protocol led to poor repeatability. In this study, Nannochloropsis gaditana NIES-2587 was cultured in f/2 liquid medium. The culture growth was estimated on optical density value and the lysis process was improved using gradual temperature procedure during the PCR process. The same culture was extracted using manual DNA extraction method for comparison. The DNA obtained from both methods were amplified using RbclN primer pair to amplify 1486 bp partial sequence of Nannochloropsis rbcL gene, followed by the sequencing of the PCR product. Molecular identification based on the sequence result and BLAST analysis indicated that direct PCR and manual DNA extraction methods successfully produced high sequences result and confirmed the identity of microalgae species into N. gaditana strain CCMP527 with a genetic similarity of >99%.

Rapid and Convenient Quantitative Analysis of SARS-CoV-2 RNA in Serous Saliva with a Direct PCR Method.

Sensitive and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), frequently performed using direct polymerase chain reaction (PCR), is essential for restricting the spread of coronavirus disease 2019 (COVID-19). However, studies evaluating accurate detection are still required. This study evaluated the quantitativeness and sensitivity of the Ampdirect™ 2019-nCoV detection kit, a direct PCR method. Using saliva with or without Tris-buffered saline (TBS) dilution, linearity, and limits of the N1 and N2 regions of SARS-CoV-2 genomic RNA were assessed using EDX SARS-CoV-2 RNA standard dissolved in RNase-free water (RFW). Fluorescence intensities in non-diluted saliva were higher than those in TBS-diluted samples. Linear regression analysis of detected quantification cycle values and spiked standard RNA concentrations showed that the coefficient of determination of the N1 and N2 genes was 0.972 and 0.615 in RFW and 0.947 and 0.660 in saliva, respectively. N1- and N2-positive detection rates in saliva were 46% (6/13 tests) and 0% (0/12 tests) at one copy/reaction, respectively. These results indicate good quantitativeness and sensitivity for N1 but not for N2. Therefore, our findings reveal that the Ampdirect™ 2019-nCoV system, especially targeting the N1 gene, enables rapid and convenient quantification of SARS-CoV-2 RNA in saliva at one copy/reaction.

Practical use of multiplex and broad-range PCR in ophthalmology.

Polymerase chain reaction (PCR) can be used to confirm or deny infectious ocular inflammation such as uveitis. The purpose of this article is to review the current practical use of PCR examination in ophthalmology, especially multiplex and broad-range PCR, and a novel PCR, termed Strip PCR. At first, in the Introduction, we show the development of the PCR examination in ophthalmology. We next show the clinical applications of multiplex PCR and broad-range PCR. These advances in PCR continue to contribute greatly to the ophthalmology field. We also show how the sample for PCR is collected. Recently, we established a novel examination, a multiplex real-time PCR (Strip PCR) prototype for detecting 24 pathogens responsible for ocular infectious diseases. Moreover, we developed the Direct Strip PCR method, which skips the DNA extraction step in the procedure. This PCR is anticipated to ease etiologic evaluation, increasing pathogen detection in the intraocular fluids of uveitis patients even by general ophthalmologists. We also describe the following: (1) representative cases in which PCR is useful, (2) representative cases in which PCR can exclude a diagnosis, (3) the current status of PCR in the diagnosis of infectious uveitis and advanced medical service, and (4) the prospects for clinical PCR in ophthalmology. We have established and developed the multiplex PCR, broad-range PCR, Strip PCR, and Direct Strip PCR methods and have reported the efficacy of such tests in multicenter studies. Our goal is "rapid and simple comprehensive PCR diagnosis anywhere and by anyone" for ocular infections.

A quick method for obtaining high-quality DNA barcodes without DNA extraction in microalgae

The identification of microalgae based on their morphological features is always challenging due to their similar traits. DNA barcoding is an established technique for obtaining information about genomic sequences to identify different species. However, obtaining DNA from microalgae requires long cultivation times in order to produce enough cells and many steps are needed for the extraction of DNA, requiring significant time and money. In previous studies direct polymerase chain reaction (PCR) has been suggested as a feasible approach for some microalgae, but this protocol still requires pretreatment to deal with the specimens. In the current study, a direct PCR that avoids the DNA extraction step was tested in Chlorella, Scenedesmus, Dunaliella salina and diatoms living in freshwater and seawater. Our optimization involves the following three major aspects: (1) the use of different species, (2) protocol simplification, and (3) microalgae and DNA amount. We also detected DNA levels required for our direct PCR method. After the optimization, the improvement realized was our method’s simplicity, efficiency, and cost-effectiveness, since all the pretreatments were omitted. However, no alterations were observed in the results and equal quality PCR products were obtained compared with conventional PCR. Also, this technique has the same phylogenetic utility as the other PCR methods. To the best of our knowledge, this is the simplest and most direct way to perform PCR in both freshwater and marine microalgae. We also show that the optimized results can be used for routine multi-locus phylogenetic analysis and demonstrate high resolution of DNA barcoding gaps.

Isolation and In Vitro Screening of Lactic Acid Bacteria from Some Local Brewed Drinks, Rotten Fruits, Pig Hindgut and their Characterization

The aim of this study was to screen, isolate and characterize Lactic acid bacteria from brewed drinks, certain rotten fruits and hindguts of pigs, using biochemical and Polymerase Chain Reaction (PCR) methods and to compare them with patented probiotics. Briefly, 200 ml each of Local brews kunu zaki, nono and burukutu were purchased separately from five different points and later pooled to form one liter each. Near rotten pineapples (17) and oranges (3) were purchased, and segments of the hindgut (cecum, colon and rectum) of six slaughtered pigs were collected. Bacteria growths were identified according to their morphological and biochemical characteristics. An in vitro fermentation of four grains, namely: sorghum ( Sorghum bicolor ) yellow and red varieties, Millet ( Pennisetum americanum ) and Maize ( Zae mays ) were later performed using the isolated Lactobacillus bacteria from the local brews, fruits and pig hindgut and compared with the patented bacteria. This process was repeated with 0.01ml of an over - night culture of MRS broth concentration containin g one of the two 10 5 and 10 7 Cfu/ml Lab isolate from local drinks, fruits and pig hindgut isolates. Selected fermenters from pig hindgut and burukutu successfully decreased pH of Millet, Maize and Sorghum from 6 to 3.80 and from 6 to 3.92 respectively henc e can be used as fermenters. Molecular confirmation of Lactobacillus species in pig hind gut and burukutu using direct PCR method and the amplification of the genomic DNA showed that the genus level of Lactobacillus was the same for all the isolates and th e homology analysis inferred from the 16S RNA sequence clearly verified that all the strains were Lactobacillus species. L. casei and L. acidiphilus were identified as species type from burukutu . But none from pig hind gut. Therefore it was concluded that Millet, Maize and Sorghum can be effectively used as components of fermented pig diets and that local brewed drink; burukutu and pig hind gut content are good sources of Lactobacillus bacteria f or fermentation of feed for feeding pigs.

Screening of crows and waterfowls for Salmonella and Listeria monocytogenes infection

Background and Aim: Wild birds can carry a number of potential human and animal pathogens. These birds can intrude into human habitats giving the opportunity to transmission of such infection. Therefore, the current study was designed to investigate the role of crows and waterfowls as vectors of Salmonella and Listeria monocytogenes and to compare cultivation methods with direct polymerase chain reaction (PCR) for the detection of infection. Materials and Methods: A total of 200 fecal dropping samples were collected. Salmonella was cultivated by three step method (Universal Pre-enrichment Broth [UPB], Rappaport-Vassiliadis Broth, and Xylose Lysine Desoxycholate agar). The recovered isolates were characterized by biotyping, serotyping, and PCR detection of enterotoxin (stn) gene. The antibiogram pattern of isolates against a panel of 8 antibiotics was recorded. L. monocytogenes was cultivated on UPB, then on Listeria Oxford Agar and Listeria CHROMagar. The recovered isolates were characterized by biotyping and PCR detection of listeriolysin (hylA) gene. Results: The percentages of Salmonella infection in crows and waterfowls were 10 and 20, respectively. The most frequently recovered serovars were Typhimurium, Potengi, Enteritidis, and Kentucky. Antimicrobial susceptibility analysis of Salmonella isolates showed that the resistance rates for gentamicin were the highest (92%), followed by amoxicillin (88%) and cefixime (60%). Resistance to 8 antibiotics was recorded in 60% (15/25) of Salmonella isolates. The percentages of L. monocytogenes infection in crows and waterfowls were 1.3 and 2, respectively. Direct PCR applied to UPB revealed that 12% and 3% of samples were positive for Salmonella and L. monocytogenes, respectively, and there was no significant difference between direct PCR and cultivation method for the detection of infection. Conclusion: The present findings indicate that wild birds can harbor zoonotic enteric pathogens and this necessitates monitoring the epidemiologic status of these pathogens among wild birds and humans and applying the appropriate intervention measures to prevent the spread of infection.

A thaumatin-like genomic sequence identification in Vitis vinifera l., stormy wines and musts based on direct pcr

Direct polymerase chain reaction method was use to amplify a thaumatin-like sequence of Vitis vinifera L. in grapes as well as in stormy wines and musts. Thaumatin-like proteins (TLPs) of Vitis vinifera possess beside its function in abiotic and biotic stress response another one - they are able to cause protein haze in wine unless removed prior to bottling. Direct PCR is an approach where omission of DNA extraction is typical prior the amplification of the target site of plant genome. Crude extract or small pieces of plant tissues are used in the analysis directly without steps of extraction and purification of gDNA. The biological material that was used in analysis was collected during August - October 2017 in local stores and winery Sabo and comprises from cultivars Iršai, Muškát, Savignon Blanc, Svätovavrinecké, Dornfelder and Pálava. Direct PCR was performed by a cutted piece of grape tissue and a dilution buffer was use in 1:2 for stormy wine or must, respectively. Direct amplification of thaumatin-like protein sequence of Vitis vinifera was performed along with the control reactions with the primers for conserved region of plant chloroplast. Possitive amplification of thaumatin-like allergen sequence resulted in 570 bp amplicon. The most abundant amplicons were amplified in stormy wines, followed by musts and the amplicons from grapes were weaker when comparing them to others. The amplicon specificity checking of obtained PCR product of thaumatin-like allergen was performed by restriction cleavage by Psi I and resulted in restriction amplicons of the 80 bp, 81 bp, 94 bp and 315 bp in length. Confirmation of the amplicon specificity by restriction cleavage support the potential of direct PCR to become a reproducible method that will be fully applicable in routine analysis of not only plant genomes in the future, but it was demonstrated, that it works in liquids, too.

Simplifying Nucleic Acid Amplification from Whole Blood with Direct Polymerase Chain Reaction on Chitosan Microparticles.

Tremendous advances have been made in the development of portable nucleic acid amplification devices for near-patient use. However, the true limitation in the realization of nucleic acid amplification tests (NAATs) for near-patient applications is not the amplification reaction, it is the complexity of the sample preparation. Conventional approaches require several precise intervention steps during the protocol. There are numerous reports in the literature that mimic the sample preparation procedure within a lab-on-a-chip device or cartridge, but these systems require a high number of integrated steps, making the devices and/or their supporting equipment too complex to meet the necessary cost targets and regulatory requirements for near-patient applications. Here we report a simplified method to purify and amplify DNA from complex samples in a minimal number of steps. We show that chitosan-coated microparticles can lyse human cells and capture the released DNA in a single mechanical agitation step, and we show that bound DNA can be amplified directly from the microparticle surface when the magnetic microparticles are transferred to a polymerase chain reaction (PCR). This procedure eliminates (i) the use of PCR-inhibiting reagents (e.g., chaotropic salts and alcohol) and (ii) the washing and elution steps that are required to remove these reagents and release DNA in typical NAAT sample preparation methods. To illustrate the use of this direct PCR method in diagnostics, we amplify human genomic DNA sequences from a ∼1 μL droplet of whole blood, and we amplify plasmid DNA spiked into whole blood droplets to represent circulating viral DNA or cell-free DNA. The qPCR threshold cycle for direct PCR from whole blood is comparable to that of direct PCR with purified DNA, demonstrating that the lysis and capture steps effectively bind DNA and sufficiently enable its amplification. Furthermore, the efficient amplification of plasmid DNA spiked into whole blood proves that the large mass of human genomic DNA captured from the lysed cells does not inhibit the capture and amplification of other circulating DNA. We anticipate that this new streamlined method for preparing DNA for amplification will expand the diagnostic applications of nucleic acid amplification tests, in particular for near-patient applications.

Role of common sarcomeric gene polymorphisms in genetic susceptibility to left ventricular dysfunction.

Mutations in sarcomeric genes are common genetic cause of cardiomyopathies. An intronic 25-bp deletion in cardiac myosin binding protein C (MYBPC3) at 3' region is associated with dilated and hypertrophic cardiomyopathies in Southeast Asia. However, the frequency of sarcomeric gene polymorphisms and associated clinical presentation have not been established with left ventricular dysfunction (LVD). Therefore, the aim of the present study was to explore the association of MYBPC3 25-bp deletion, titin (TTN) 18 bp I/D, troponin T type 2 (TNNT2) 5 bp I/D and myospryn K2906N polymorphisms with LVD. This study includes 988 consecutive patients with angiographically confirmed coronary artery disease (CAD) and 300 healthy controls. Among the 988 CAD patients, 253 with reduced left ventricle ejection fraction (LVEF≤45%) were categorized as LVD. MYBPC3 25-bp deletion, TTN 18 bp I/D and TNNT2 5 bp I/D polymorphisms were determined by direct polymerase chain reaction method, while myospryn K2906N polymorphism by TaqMan assay. Our results showed that MYBPC3 25-bp deletion polymorphism was significantly associated with elevated risk of LVD (LVEF <45) (healthy controls versus LVD: OR=3.85, P <0.001; and nonLVD versus LVD: OR=1.65, P = 0.035), while TTN 18 bp I/D, TNNT2 5 bp I/D and myospryn K2906N polymorphisms did not show any significant association with LVD. The results also showed that MYBPC3 25-bp deletion polymorphism was significantly associated with other parameters of LV remodelling, i.e. LV dimensions (LV end diastole dimension, LVEDD: P = 0.037 and LV end systolic dimension, LVESD: P = 0.032). Our data suggests that MYBPC3 25-bp deletion may play significant role in conferring LVD as well as CAD risk in north Indian population.

Detection of short tandem repeat polymorphisms from human nails using direct polymerase chain reaction method.

Human nail is an important forensic material for parental testing and individual identification in large-scale disasters. Detection of STR polymorphism from hard tissues generally requires DNA purification, which is technically complicated and time consuming. In the present study, we attempted to detect STR polymorphisms from untreated human nail samples by direct PCR amplification method using the primer mixture supplied with the GenePrint® SilverSTR® III System or the AmpFℓSTR® Identifiler® PCR Amplification Kit, and Tks Gflex DNA polymerase known to be effective for amplification from crude samples. A nail fragment measuring approximately 1.5 mm in breadth and 0.5 mm in length was placed directly into a PCR tube, and various PCR conditions were tested. The PCR products were analyzed by denaturing acrylamide gel electrophoresis or CE. Multiple STR polymorphisms were detected successfully. This method that detects STR polymorphisms not only from fresh human fingernails, but also from old nail fragments stored at room temperature for up to 10 years is expected to become a novel DNA analytical method in forensic medicine and genetic studies.

Convenient detection of the citrus greening (huanglongbing) bacterium 'Candidatus Liberibacter asiaticus' by direct PCR from the midrib extract.

A phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’ (Las) is a major pathogen of citrus greening (huanglongbing), one of the most destructive citrus diseases worldwide. The rapid identification and culling of infected trees and budwoods in quarantine are the most important control measures. DNA amplification including conventional polymerase chain reaction (PCR) has commonly been used for rapid detection and identification. However, long and laborious procedures for DNA extraction have greatly reduced the applicability of this method. In this study, we found that the Las bacterial cells in the midribs of infected leaves were extracted rapidly and easily by pulverization and centrifugation with mini homogenization tubes. We also found that the Las bacterial cells in the midrib extract were suitable for highly sensitive direct PCR. The performance of direct PCR using this extraction method was not inferior to that of conventional PCR. Thus, the direct PCR method described herein is characterized by its simplicity, sensitivity, and robustness, and is applicable to quarantine testing.

콩 종자에서 Xanthomonas axonopodis pv. glycines의 검출을 위한 Direct PCR 방법 개발

콩 불마름병을 일으키는 Xanthomonas axonopodis pv. glycines를 종자에서 DNA 추출없이 바로 검출하는 방법에 대하여 연구하였다. 콩 종자에서 X. axonopodis pv. glycines를 특이적으로 검출하기 위해 특이적인 유전자로 알려져 있는 glycinecin A로부터 증폭 size가 401 bp인 primer Xag F1 & Xag R1을 고안하였다. Xag Fl과 Xag R1 primer는 콩 종자와 잎에서 분리한 균주와 KACC에서 분양받은 X. axonopoids pv. glycines 균주를 증폭시켰으나 근연종인 X. axonopodis pv. citri, X. axonopodis pv. vesicatoria 등은 증폭되지 않았다. 콩 종자에 존재하는 것으로 알려진 다른 세균들 역시 증폭되지 않았다. 고안된 Xag F1 & Xag R1 primer를 이용한 X. axonopodis pv. glycines의 검출 한계 농도 측정은 genomic DNA와 세포 현탁액을 이용하였다. X. axonopodis pv. glycines의 genomic DNA는 200 fg까지 증폭이 되었으며, 세포현탁액은 <TEX>$OD_{600nm}$</TEX> 0.1로 농도를 조정한 뒤, <TEX>$10^{-8}$</TEX>까지 희석하여 측정한 결과 <TEX>$10^{-6}$</TEX>인 <TEX>$1.8{\times}10^3$</TEX> cfu/ml까지 증폭이 확인되었다. 자연 감염된 종자에서 병원균을 검출하기 위해 종자를 육안상 건전종자와 불건전한 종자(변색립, 피해립)로 구분하여 direct PCR 방법으로 실험 한 결과 육안상 건전종자에서는 병원균이 검출되지 않았으나 불건전한 종자에서는 병원균의 검출이 확인되었다. 또한 진탕 배양 시간에 따른 병원균의 검출여부를 조사한 결과 진탕 배양 2시간부터 병원균의 검출이 확인되었으며 시간이 지날수록 증폭 밴드가 더욱 선명해 지는 것을 확인할 수 있었다. 그러므로 고안된 Xag Fl & Xag R1 primer를 이용한 direct PCR 방법은 다른 많은 미생물들로 오염되어진 콩종자에 있는 X. axonopodis pv. glycines를 신속하고 민감하게 검정할 수 있는 효과적인 방법으로 활용될 수 있을 것이다. Direct Polymerase Chain Reaction (PCR) method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Xanthomonas axonopodis pv. glycines on soybeen seeds without DNA isolation. Primers Xag F1 and Xag R1 were designed to specifically amplify a 401 bp fragment of the glycinecin A gene of X axonopodis pv. glycines. Xag F1 and Xag R1 were used to carry out the PCR analysis with genomic DNA from 45 different bacterial strains including phylogenetically related bacteria with X axonopodis pv. glycines, and other bacterial strains of different genus and species. The PCR assay using this set of primers were able to detect X axonopodis pv. glycines with DNA concentration as low as 200 fg and <TEX>$1.8{\times}10^3$</TEX> cfu/ml. The Xag was detected from the seed samples incubated for 2 hrs with shaking and the intensity of the band was increase with the incubation time of seeds. The Direct PCR assay method without DNA isolation makes detection of X. axonopodis pv. glycines on soybean seeds easier and more sensitive than other conventional methods. The developed seed assay using direct PCR method will be useful for the specific detection of X. axonopodis pv. glycines in soybean seed samples.

A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis

A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli beta-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification.

Optimization of Sample Size and DNA Extraction Methods to Improve PCR Detection of Different Propagules of Phytophthora infestans.

The plant pathogen Phytophthora infestans causes a destructive blight of potato tubers and foliage. A rapid polymerase chain reaction (PCR) assay has been developed for detection of P. infestans in potato tubers. In this study, the effect of method of DNA extraction on different propagule types and the minimal number of propagules of P. infestans detectable by PCR were assessed using the PINF and internal transcribed spacer (ITS)5 primers. Sensitivity of the primers for PCR was high, and DNA was detectable at concentrations as low as 10 pg/ml. Zoospores and oospores responded differently to different extraction methods, whereas all extraction methods worked equally well for sporangia. Freeze-thaw DNA lysis, in which propagules were frozen at -80°C and thawed at 65°C three times for 15 min each, or direct PCR, in which propagules were placed directly in the reaction mix, were effective methods for PCR detection of sporangia or zoospores but were not effective methods for PCR detection of DNA in oospores of P. infestans. DNA from a single sporangium or oospore could be amplified by PCR after hexadecyltrimethyl-ammonium bromide (CTAB) or NaOH lysis extraction methods, whereas DNA from a single zoospore could be amplified by CTAB or direct PCR methods. "IsoCode" Stixs, used in forensic applications, were used to collect the pathogen from leaf and tuber lesions and provided another simple method to extract template DNA. PCR detection of the pathogen in infected tubers using PINF and ITS5 primers was compared to tissue isolation or visual observation. The probability of detection of P. infestans in infected tubers at 7 days post inoculation using the PCR assay, tissue isolation, or visual observation was 0.90, 0.80, and 0.75, respectively. The PINF and ITS5 primers provide a powerful tool for rapid and sensitive detection of zoospores, sporangia, and oospores of P. infestans when used with appropriate extraction methods, and could easily be deployed to reduce spread of the pathogen in potato tubers.

ＰＣＲ法による食肉およびふん便中の下痢原性大腸菌の迅速検査法の検討

A direct polymerase chain reaction method to EC cultured broth (named EC-PCR) for screening detection of enteropathogenic E. coli (ETEC, VTEC and EIEC) in foods and feces is described.Eleven E. coli strains belonging to three groups (4ETEC: 06, 025, 0128, 0148; 4VTEC: 026, 0111, 0145, 0157; and 3EIEC: 028ac, 0124, 0164) were used for this study. Four pairs of oligonucleotide primers homologous to LT, ST, VT and EIEC genes were used in combination. The culture condition at 37-43°C for 16-20 h in EC broth was most suitable for the EC-PCR method, and no cross readings were observed with other bacteria, substances in meats or feces.After a 20-h enrichment step, it was possible to detect fewer than 10 to 102 bacteria per g of the aritificially inoculated meat. E. coli was easily detected because of low contamination with other bacteria which disturb the detection of E. coli. However, in feces, it ranged from 103 to 104 cfu per g. The large number of bacteria with feces are the main limiting factor of the EC-PCR detection assay. All E. coli strains examined were detected from all the enrichment cultures on both the EC-PCR and the culture methods. In two sporadic cases and two food poisoning cases, the enteropathogenicity of E. coli isolates from patients was rapidly judged by the EC-PCR method.These findings were consistent with those of the culture method. Thus, the findings suggest that the EC-PCR method is a suitable, sensitive and rapid method for detection of the potentially enteropathogenic E. coli.

Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification.

A SpeI-AvaI fragment (0.3 kbp) from pBbi16 (a pBR322 derivative containing a 6.3-kbp Babesia bigemina DNA insert) was subcloned into the pBluescript phagemid vector and was sequenced by the dideoxy-mediated chain termination method. Two sets of primers were designed for the polymerase chain reaction (PCR) assay. Primer set IA-IB was used to amplify a 278-bp DNA fragment, and primer set IAN-IBN was used to prepare a probe directed to a site within the PCR-amplified target DNA. Digoxigenin-dUTP was incorporated into the probe during the amplification reaction. PCR amplification of target DNA obtained from in vitro-cultured B. bigemina and nucleic acid hybridization of amplified product with the nonradioactive DNA probe showed that a 278-bp fragment could be detected when as little as 100 fg of parasite genomic DNA was used in the assay. A fragment of similar size was amplified from genomic DNAs from several B. bigemina isolates but not from DNAs from Babesia bovis, Anaplasma marginale, or six species of bacteria or bovine leukocytes. Similarly, the PCR product could be detected in DNA samples purified from 200 microliters of blood with a parasitemia of as low as 1 in 10(8) cells and which contained an estimated 30 B. bigemina-infected erythrocytes. By a direct PCR method, B. bigemina DNA was amplified from 20 microliters of packed erythrocytes with a calculated parasitemia of 1 in 10(9) cells. With the analytical sensitivity level of the PCR-DNA probe assay, six cattle with inapparent, 11-month chronic B. bigemina infection were found to be positive. No PCR product was observed in bovine blood samples collected from a splenectomized, A. marginale-infected bovine, a 4-year chronic B. bovis-infected animal, or 20 uninfected cattle from Missouri which were subjected to amplification. The PCR-DNA probe assay was shown to be sensitive in detecting latently infected cattle. The specificity and high analytical sensitivity of the test provide valuable tools for performing large-scale epidemiological studies.