A non-stop, single-tube, semi-nested PCR technique for grading the severity of white spot syndrome virus infections in Penaeus monodon.

A single-tube, non-stop, semi-nested polymerase chain reaction (PCR) technique was developed for simultaneous detection and severity grading of white spot syndrome virus (WSSV) infections in the black tiger shrimp Penaeus monodon. The test uses 1 sense primer and 3 antisense primers that produce up to 3 PCR products (1100, 526 and 250 base pairs [bp]) depending upon the severity of infection. Specifically, heavy infections (> or = 2 x 10(4) viral particles) of WSSV produce all 3 fragments, while moderate infections (around 2 x 10(3) viral particles) produce 2 (526 and 250 bp) and light infections (20 to 200 viral particles) produce 1 (250 bp). In addition, the technique uses internal control primers that yield a shrimp characteristic fragment for non-infected samples and samples with a low quantity of viral target in order to assure integrity and reproducibility of the PCR assays. The non-stop, single-tube, semi-nested PCR technique is simple and convenient and can detect as little as 5 fg WSSV DNA (20 viral particles) in crude extracts of postlarval samples or extracts of pleopods and haemolymph from larger shrimp.     

Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction

A 501 bp caf1 gene fragment and a 443 bp of pla gene fragment carried by 100 kb (pFra) and 10 kb (pPst) species-specific extrachromosomal replicons, respectively, were used as targets to study the conditions under which DNA amplification by polymerase chain reaction (PCR) may be applied to detect and identify Yersinia pestis DNA in cell lysates of pure cultures and biological samples. The sensitivity limit of PCR with the crude cell lysates of Y. pestis EV was estimated as 10–50 cfu in reaction mixture. When target Y. pestis EV cells were mixed with fresh blood of white mice, which contained 0.4% potassium citrate, the PCR detection level varied from 400 to 100 cfu ml^-1 of blood depending on the method used for preparing the sample. In our tests PCR was effective for the detection of yersinia in the blood of white laboratory mice experimentally infected with virulent Y. pestis KM638 strain. This method can be considered convenient for routine detection and identification of Y. pestis.     

Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution

We examined whether crude DNA extracts prepared from gene-engineered mouse tissues are suitable as a template for zygosity determination by SYBR Green real-time genomic PCR. A crude DNA solution was prepared by brief incubation with lysis buffer containing ear, tail, or fetus of ROSA26 mouse, a gene-trapped strain carrying the β-galactosidase (β-gal) gene. Five serially diluted crude DNA samples (original, 2-, 4-, 8-, 16-diluted) were next prepared and then subjected to three-step (95°C, 60°C and 72°C) reactions of real-time PCR to detect the β-gal gene and the receptor-activity-modifying protein 3 (ramp3) gene (as an internal reference gene). The slopes of standard curves obtained from the real-time PCR indicated that amplification efficiency was approximately 99%, and the efficiencies of target and reference were almost equal. With this system, we next determined the zygosity of mice derived from mating heterozygous ROSA26 females and males, and found a sharp distinction in zygosity, wild-type, heterozygous and homozygous. Assessment of crude DNA samples from other gene-engineered mice including B6ZP3Cre-Tg, B6rAM-Tg, and Ramp2-gene-targeted strains revealed that our method was effective for determination of zygosity. The present method is more convenient and rapid than formerly published methods employing purified genomic DNA as a template. Our method will be particularly useful for experiments requiring rapid and accurate genotyping of gene-modified animals/fetuses.     

Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp

A real-time PCR method was developed to detect monodon baculovirus (MBV) in penaeid shrimp. A pair of MBV primers to amplify a 135 bp DNA fragment and a TaqMan probe were developed. The primers and TaqMan probe were specific for MBV and did not cross react with Hepatopancreatic parvovirus (HPV), White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic virus (IHHNV) and specific pathogen free (SPF) shrimp DNA. A plasmid (pMBV) containing the target MBV sequence was constructed and used for determination of the sensitivity of the real-time PCR. This real-time PCR assay had a detection limit of one plasmid MBV DNA copy. Most significantly, this real-time PCR method can detect MBV positive samples from different geographic locations in the University of Arizona collection, including Thailand and Indonesia collected over a 13-year period.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

DNase I treated DNA-PCR based detection of food pathogens immobilized by metal hydroxides

Bacterial immobilization by metal hydroxides can be used for enrichment of various bacterial strains including Gram (+) and Gram (−). The polymerase chain reaction (PCR)-based bacterial detection without enrichment culture could be implemented by concentrating bacteria from food matrix by metal hydroxides. To distinguish between viable and non-viable cells, it is often required to detect the mRNA, an indicator of viable cells. This technique, although provides accurate and reliable result, is expensive and time-consuming. Here, we report the studies on application of DNase I treatment to eliminate DNA from dead cells and subsequently detect the presence of viable pathogens by conventional PCR. It was found that treatment of immobilized cells with DNase I for 1 h at 37°C prior to DNA extraction could efficiently eliminate false positives due to the presence of non-viable cells. The technique was used to detect the presence of various pathogens in food model. The detection limits for Escherichia coli O157:H7 (384 bp), Listeria monocytogenes (482 bp), and E. coli wild type (580 bp) was 5 × 10¹ cells and that for Salmonella typhimurium (685 bp) was 5 × 10² cells in 10 ml of whole milk. An erratum to this article can be found at     

Development of PCR primers from internal transcribed spacer region 2 for detection of Phytophthora species infecting potatoes.

We developed PCR primers and assay methods to detect and differentiate three Phytophthora species which infect potatoes and cause late blight (Phytophthora infestans) and pink rot (P. erythroseptica and P. nicotianae) diseases. Primers based on sequence analysis of internal transcribed spacer region 2 of ribosomal DNA produced PCR products of 456 bp (P. infestans), 136 bp (P. erythroseptica), and 455 bp (P. nicotianae) and were used to detect the pathogens in potato leaf (P. infestans) and tuber (P. infestans, P. erythroseptica, and P. nicotianae) tissue with a sensitivity of 1 to 10 pg of DNA. Leaf and tuber tissue were processed for PCR by a rapid NaOH method as well as a method based on the use of commercially available ion-exchange columns of P. infestans primers and the rapid NaOH extraction method were used to detect late blight in artificially and naturally infected tubers of potato cultivar Red LaSoda. In sampling studies, P. infestans was detected by PCR from artificially infected tubers at 4 days postinoculation, before any visible symptoms were present. The PCR assay and direct tissue extraction methods provide tools which may be used to detect Phytophthora pathogens in potato seedlots and storages and thus limit the transmission and spread of new, aggressive strains of P. infestans in U.S. potato-growing regions.     

Analysis of Diverse Plant Species using Mass Spectrometric Cleaved Amplified Polymorphic Sequences (MS-CAPS) and Improvement of PCR Efficiency by Addition of Cysteine

The applicability of mass spectrometric cleaved amplified polymorphic sequences (MS-CAPS) was evaluated in several plant species. This method consists of genomic DNA extraction from plant tissues, polymerase chain reaction (PCR) amplification of a specific genetic region, enzymatic digestion of amplicons, and followed by rapid analysis of single nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Crude extracts obtained by homogenizing plant tissues in water were used as templates for short PCR amplifications for MS-CAPS analysis. For most plant species tested, these crude extracts could be used directly as templates for PCR. However, extracts from lettuce leaves and stems showed enzymatic browning as a result of polyphenol oxidase (PPO) activity, and were not suitable PCR templates. The addition of cysteine to the homogenizing solution inhibited enzymatic browning and did not affect the other MS-CAPS procedures, including PCR amplification, uracil-DNA glycosylase treatments, or MALDI-TOF MS analysis. Thus, this method inhibits PPO in crude extracts, allowing them to be used directly for MS-CAPS analysis.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

Direct-PCR Detection and Epidemiology of Ramularia collo-cygni Associated with Barley Necrotic Leaf Spots

Ramularia collo‐cygni (Sutton & Waller) is involved in a disease complex of barley characterized by the formation of necrotic spots on leaves. Isolation of this fungus is difficult, which complicates the study of its epidemiology and the aetiology of the disease complex. A new assay based on polymerase chain reaction (PCR) was developed to detect the presence of R. collo‐cygni (Rcc) without previous isolation of the fungus nor prior purification of DNA. Primers RC3 and RC5 were designed to amplify a 348 bp fragment of the internal transcribed spacer region of this pathogen. These primers were highly specific to Rcc, as no cross‐reactions were observed with other barley pathogens or saprobes commonly found on this crop. Amplification was possible from crude extracts (direct‐PCR), circumventing the need for a DNA purification. Detection of 1 fg of target DNA was achieved with a single PCR. This specific and sensitive assay was used to study the epidemiology of Rcc on winter and spring barley at two locations in Switzerland. Rcc was present on winter barley from snow melting until harvest and colonized gradually all leaf layers. It was also found on volunteers, which could, together with weeds, offer a survival possibility to the pathogen between two barley crops. The fungus was detected on spring barley only after it sporulated on winter barley, indicating that Rcc probably spreads from winter to spring barley. The hypothesis of Rcc being a seed‐borne pathogen can be tested more easily with this fast and reliable molecular tool, which may also find applications in barley breeding programmes and fungicide trials.     

Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization,dot blot hybridization and PCR amplification

Hepatopancreatic parvovirus (HPV) infects the hepatopancreas in penaeid shrimp and retards their growth. The DNA sequence of HPV from Thai shrimp Penaeus monodon (HPVmon) differs from HPV of Penaeus chinensis (HPVchin) by approximately 30%. In spite of this difference, commercial PCR primers (DiagXotics) developed from HPVchin to yield a 350 bp PCR product do give a 732 bp product with HPVmon DNA template. On the other hand, the sensitivity of HPVmon detection with these primers and with hybridization probes designed for HPVchin is significantly lower than it is with HPVchin. To improve sensitivity for HPVmon detection, we used the sequence of the 732 bp HPVmon PCR amplicon described above to develop specific PCR primers (H441F and H441R) and hybridization probe. The primers could detect as little as 1 fg of purified HPVmon DNA while the 441 bp digoxygenin-labeled PCR product gave strong, specific reactions with in situ hybridization and with hybridization blots. In contrast, negative results were obtained using DNA from all other pathogens tested and from DNA of P. monodon. Supernatant solution from boiled, fresh shrimp fecal and postlarval samples homogenized in 0.025% NaOH/0.0125% SDS could be used to detect as little as 0.1 pg HPVmon DNA by the PCR reaction. By dot blot hybridization, a visible signal was obtained with purified HPVmon DNA at 0.01 pg, but detection in spiked feces and postlarval samples was only 1 and 0.1 pg, respectively.     

Development of a simple PCR-based assay for the identification of triazine resistance in the noxious plant common ragweed (Ambrosia artemisiifolia) and its applicability in higher plants

Bidirectional allele-specific PCR (Bi-PASA) was used to detect a point mutation causing triazine resistance in common ragweed (Ambrosia artemisiifolia). The external primers amplified a 278 bp standard DNA fragment in all genotypes. In the susceptible S264S genotypes, a 208 bp fragment was expected while in resistant S264G common ragweed genotypes a 109 bp band was expected. In resistant plants, both the wild and mutant type fragments were detected, indicating that the original triazine sensitive cpDNA is maintained in a heteroplasmic state in the resistant S264G genotypes. Additionally, in silico analysis confirmed the potential applicability of our diagnostic assay for other plant species. In 24 out of 74 taxa (32%), the assay could be used without any change, while in the others some of the primers should be redesigned before use.     

A case of mistaken identity? Vaccinia virus in a live camelpox vaccine

Live-attenuated (LA), and inactivated adjuvant (IA) camelpox virus (CMLV) vaccines are produced in several countries worldwide. A tissue culture attenuated CMLV isolated (Jouf-78) is used to produce an LA vaccine in Saudi Arabia (Hafez et al., 1992). DNA extracts from the Saudi LA vaccine were used as positive controls for a routine ATIP PCR produced fragments longer than 881 bp. PCR-amplified ATIP sequences were similar to vaccinia virus (VACV) Lister strain. PCR and sequence analysis of two extracellular enveloped virus (EEV)-specific (A33R and B5R), and two intracellular mature virus (IMV) (L1R and A27L) othrologue genes from the vaccine DNA extracts confirmed the finding. CMLV sequences were not detected in vaccine DNA extracts. A VACV Lister strain imported from Switzerland was used in control experiments during initial testing of the Saudi LA vaccine. High antigenic similarity between VACV and CMLV, and a possible contamination event during production may have caused this issue. Environmental and health impact studies were recommended because early VACV vaccines produced in some European countries contained nonhighly attenuated strains that were not adequately screened for adventitious agents.     

Detection of Escherichia coli in sewage and sludge by polymerase chain reaction.

A method in which the polymerase chain reaction (PCR) was used was developed to amplify either a uidA gene fragment or a 16S rRNA gene fragment from Escherichia coli in sewage and sludge. Because of interference caused by humic acidlike substances, crude DNA extracts were purified with a Sephadex G-200 spun column before the PCR was begun. A Southern analysis in which a nonradioactive chemiluminescent method was used was performed to confirm the presence of PCR products. The sensitivity of detection for PCR products when the chemiluminescent method was used was determined to be 30 ag of E. coli genomic DNA template. In seeded sludge, the PCR amplified the target DNA from 80 E. coli cells per g of sludge and 50 Shigella dysenteriae cells per g of sludge. Because only 0.05 aliquot of a sludge extract was used for the PCR, we deduced that the PCR detected target DNA equivalent to the DNA of 2.5 to 4 cells in the extract. The PCR amplified the uidA fragment from diluted sewage influents and effluents containing E. coli cells. Therefore, the PCR performed with a chemiluminescent gene probe can be used to detect the presence of potentially pathogenic microorganisms in sewage and sludge. This technique can be expanded to permit direct detection of pathogenic microorganisms in water samples, thus leading to enhanced public health protection.     

Use of the polymerase chain reaction and oligonucleotide probes for the rapid detection and identification of Carnobacterium species from meat.

The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (32P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.     

The identification of Cryptosporidium species and Cryptosporidium parvum directly from whole faeces by analysis of a multiplex PCR of the 18S rRNA gene and by PCR/RFLP of the Cryptosporidium outer wall protein (COWP) gene

A multiplex polymerase chain reaction (PCR) procedure to amplify 18S rRNA gene fragments has been developed. Amplified DNA fragments of the expected size were obtained which were specific for Cryptosporidium parvum and Cryptosporidium wrairi (422 bp), Cryptosporidium baileyi (11106 bp) and Cryptosporidium muris (1346 bp). Criptosporidium parvum and C. wrairi can be distinguished using a PCR/restriction fragment length polymorphism (RFLP) analysis of the Cryptosporidium outer wall protein (COWP) gene, and these two techniques were applied to DNA extracted from whole faeces using a simple and rapid procedure. Cryptosporidium parvum DNA was detected in the faeces of 72 humans and 24 calves where cryptosporidial oocysts were demonstrated using conventional light microscopy. The specific DNA fragments were not amplified using extracts of material containing other lower eukaryotic parasites.     

Identification and differentiation of human schistosomes by polymerase chain reaction

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.     

PCR products generated from unpurified Salmonella DNA are degraded by thermostable nuclease activity

Oligonucleotide primers designed from repetitive extragenic palindromic (REP) sequences were used to PCR-amplify Salmonella DNA. Unpurified template DNA, present in crude cell extracts, yielded an essentially identical banding pattern to that arising from the use of purified DNA. However, the PCR product derived from the crude preparations did not survive storage at 4°C. This post-PCR DNA degradation, attributed to endogenous Salmonella nucleases, was inhibited by the addition of EDTA. or storage at -20°C.