Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction.

Polymerase chain reaction (PCR) amplification was used to detect human cytomegalovirus (HCMV) sequences. The fragments selected for amplification were fragments of 130 and 152 base pairs (bp) located at two opposite ends of HCMV strain AD169 EcoRI fragment D. Amplification of the 152-bp DNA was consistently greater than that of the 130-bp DNA. At the optimal Mg2+ concentration of 5 mM, specific PCR amplification of 152-bp DNA with Taq polymerase was sensitive; only one AD169-infected fibroblast cell or 0.01 pg of AD169 fragment D DNA was needed for detection. This specific amplification was also found with various clinical HCMV isolates and peripheral blood cells and urine from patients. In 37 urine samples analyzed simultaneously by PCR and by virus cultivation, identical results were found in 35 samples, while 2 scored positive only by PCR. This suggests that specific amplification of 152-bp DNA is sensitive and can be used for rapid detection of HCMV infections.     

Detection of chicken anaemia agent DNA sequences by the polymerase chain reaction

Summary A polymerase chain reaction (PCR) assay was developed for detection of chicken anaemia agent (CAA) DNA. The assay used a single set of 20-base primers complementary to sequences located in the coding regions of the CAA replicative form (RF) DNA genome at positions 485 to 504 and 1048 to 1067. The observed amplification product had the expected size of 583 bp and was confirmed to derive from CAA RF DNA by a unique Hind III restriction enzyme cleavage pattern. The amplified fragment was shown to be specific for CAA RF DNA after chemiluminescence dot blot hybridisation with a digoxigeninlabelled 25-base internal probe. The optimised PCR assay was specific for CAA and highly sensitive, being able to detect a single CAA-infected MDCC-MSB1 cell and at least 100 fg of CAA RF DNA. Preliminary results also showed that the PCR assay can detect CAA DNA in clinical specimens from chicks experimentally infected with CAA.     

Nested polymerase chain reaction and in situ hybridization for detection of nucleopolyhedrosis

A nested polymerase chain reaction (PCR) and in situ hybridization were developed for detection of baculoviruses in insects or other arthropods with nucleopolyhedrosis. The nested PCR was based on the sequences of polyhedrin genes from baculoviruses. Two sets of primers were designed, primers set, 35/36, was for the first step of amplification and yielded a product of around 680 bp, the second primer, 35-1/36-1, was designed to yield a product of around 335bp from the fragment amplified by the first primer set. The sensitivity of this two-step amplification was 100 to 1000 times higher than that of the one-step amplification by primer set (35/36). Samples which contained baculovirus DNA yielded an amplification product showing the expected DNA fragment mobility, whereas nucleic acid extracted from tissue samples of clinically healthy insects or uninfected cells showed no such DNA fragment, thereby confirming the specificity of the primers. Using the 35/36 amplicon as a probe, the PenuNPV-infected cells show positive reaction by in situ hybridization. Two-step DNA amplification and in situ hybridization with the DNA probe developed in the present paper provide effective detection and diagnostic tools for screening insects or other arthropods, especially crustacean species, crabs and shrimps, for baculovirus infections, and may be important in preventing (and/or controlling/enhancing) the infection of baculoviruses.     

PCR for specific detection of haemorrhagic enteritis virus of turkeys, an avian adenovirus.

A hexon gene based PCR was developed for specific amplification of DNA sequences from the haemorrhagic enteritis virus (HEV) of turkeys. The hexon genes of different avian adenoviruses were compared for primer construction. Two regions with low sequence homology between HEV and fowl adenovirus (FAV) hexon genes were selected for primer localisation. In correlation with the known sequence data a fragment of 1647 bp was amplified from a live vaccine and spleens of turkeys suffering from haemorrhagic enteritis (HE). All other avian adenoviruses which are able to infect turkeys, i.e. FAV and turkey adenoviruses (TAV), were negative. This is the first PCR for specific detection of HEV DNA which should be useful for rapid diagnosis and epidemiological investigations of HEV infections in turkeys.     

Nested polymerase chain reaction for detection of human immunodeficiency virus type 1 DNA in clinical specimens.

A highly sensitive two-step polymerase chain reaction (PCR) method was evaluated for detection of human immunodeficiency virus type 1 (HIV-1) DNA in clinical specimens. The product resulting from the first amplification reaction is used as the template for the second PCR with an internal (nested) primer pair. Even when starting from a single copy of HIV-1 DNA, the double PCR product was readily detected by direct visualization in ethidium bromide-stained agarose gels. Amplification of minute amounts of HIV-1 DNA was successful in a considerable excess of HIV-1 negative DNA than reported previously. All of 85 HIV-1-infected individuals were PCR-positive with at least two of the three sets of primers used, 252 of 255 amplifications allowing unambiguous visualization of a unique DNA fragment of the expected size. The two-step amplification protocol is simple and rapid and fulfills the requirements of sensitivity and specificity for use in a clinical laboratory.     

Detection and heterogeneity of herpesviruses causing Pacheco's disease in parrots

Pacheco's disease (PD) is a common, often fatal, disease of parrots. We cloned a virus isolate from a parrot that had characteristic lesions of PD. Three viral clones were partially sequenced, demonstrating that this virus was an alphaherpesvirus most closely related to the gallid herpesvirus 1. Five primer sets were developed from these sequences. The primer sets were used with PCR to screen tissues or tissue culture media suspected to contain viruses from 54 outbreaks of PD. The primer sets amplified DNA from all but one sample. Ten amplification patterns were detected, indicating that PD is caused by a genetically heterogeneous population of viruses. A single genetic variant (psittacid herpesvirus variant 1) amplified with all primer sets and was the most common virus variant (62.7%). A single primer set (23F) amplified DNA from all of the positive samples, suggesting that PCR could be used as a rapid postmortem assay for these viruses. PCR was found to be significantly more sensitive than tissue culture for the detection of psittacid herpesviruses.     

TaqMan real-time PCR assay based on DNA polymerase gene for rapid detection of Orf infection

Both conventional and real time PCR (rt-PCR) assays based on the amplification of a 103bp fragment from the DNA polymerase (DNA pol) gene (conserved, non-structural) of Orf virus (ORFV) were developed for detection and semi-quantitation of ORFV DNA from infected cell culture and clinical samples. The latter technique was based on TaqMan chemistry. The rt-PCR assay was specific and sensitive as it could detect as low as 3.5fg or 15 copies of ORFV genomic DNA. Both intra- (0.38-1.0%) and inter-assay (0.53-2.87%) variabilities of rt-PCR were within the acceptable range meaning the high efficiency and reproducibility of the assay. The rt-PCR was applied successfully to detect ORFV DNA from suspected clinical samples. Further, the assay has shown a relative diagnostic sensitivity and specificity of 100% and 93.5%, respectively, when compared to B2L gene based semi-nested PCR implying a wide potential of this rt-PCR for rapid field diagnosis of Orf in sheep and goats.     

A touchdown PCR for the differentiation of equine herpesvirus type 1 (EHV-1) field strains from the modified live vaccine strain RacH

More than 50 reference strains and field isolates of equine herpesvirus type 1 (EHV-1) were examined by a touchdown PCR. Primers for specific amplification of EHV-1 DNA were chosen from the terminal and internal repeat regions of the EHV-1 genome where the high-passaged live vaccine strain RacH displays symmetric 850 bp deletions. The positive strand and one negative strand primer were designed to encompass the deletions present in RacH, and the second negative strand primer was designed to hybridize within these deletions. Discrimination between field isolates and the vaccine strain was achieved by the generation of amplification products of different size: In all EHV-1 reference strains and field isolates, a 495 bp DNA fragment was amplified specifically, whereas a 310 bp fragment was amplified when DNA of the vaccine strain RacH was used as a template. PCR amplification was only obtained in the presence of 8–10% dimethylsulfoxide and when the primer annealing temperatures were decreased stepwise from 72°C to 60°C. Under these conditions as little as 100 fg template DNA, corresponding to about 100 genome equivalents, could be detected. The PCR assay allows fast and sensitive discrimination of the modified live vaccine strain RacH from field strains of EHV-1 since it is applicable to viral DNA extracted from organ samples and paraffin-embedded tissues. It may thus be helpful for examining the potential involvement of the RacH live vaccine strain in abortions of vaccinated mares.     

Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas' disease

Amplification of DNA sequences from the kinetoplast minicircle DNA was employed as a method for the detection and classification of small numbers of Trypanosoma cruzi cells. Two overlapping fragments from the conserved 120 bp minirepeat regions of the minicircle DNA and one fragment covering the adjacent variable regions were amplified. The minimal amount of minicircle DNA required to detect a product by hybridization with an oligonucleotide probe was 0.015 fg, which represents approximately 10 molecules or 0.1% of the minicircle DNA component of a single cell. The amplification worked equally well with kDNA from several strains of T. cruzi and did not occur with kDNA from several other kinetoplastids. kDNA recovered from less than 10 trypanosomes in whole blood could be used as a template for amplification; the presence of a several billion fold excess of human DNA had no effect on the amplification process. Schizodeme analysis by hybridization with specific oligonucleotides or by direct restriction enzyme digestion could be performed on the amplified fragments representing the minicircle conserved region or variable regions. This method should prove useful as a rapid, specific and sensitive assay for Chagas' disease in chronic patients as well as for epidemiological studies of infected animals and insects.     

Multiplex PCR for the detection of African cassava mosaic virus and East African cassava mosaic Cameroon virus in cassava

A multiplex PCR was developed for simultaneous detection of African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in cassava affected with cassava mosaic disease (CMD). One set of three primers consisting of an upstream primer common for both viruses and two down stream virus-specific primers were designed for simultaneous amplification of 368 base pair (bp) and 650 bp DNA fragments specific to the replicase gene of ACMV and EACMCV, respectively. Similarly, a second set of three primers were designed for simultaneous amplification of 540 bp and 655 bp fragments specific to the coat protein gene of EACMCV and ACMV, respectively. Primers that can amplify a 171 bp fragment of the large subunit of ribulose bisphosphate carboxylase oxygenase L were included as an internal control in these assays to determine the reliability of multiplex PCR. A simplified, cost-effective and rapid sample preparation method was adapted in place of the conventional plant DNA extraction procedure for multiplex PCR detection of ACMV and EACMCV. The method was validated using CMD-infected cassava samples obtained from farmers' fields in Nigeria. The multiplex PCR is useful for reliable assessment of the prevalence of CMBs in epidemiological studies and for crop improvement and phytosanitary programs in African countries.     

Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus

A polymerase chain reaction (PCR) amplification method was developed and evaluated to detect porcine parvovirus (PPV). A pair of 20-base primers and an oligonucleotide probe were derived from the DNA sequences common to two isolates of PPV, NADL-8 and NADL-2. The primers flanked 118-bp nucleotides within the region coding for the major structural protein VP2. After DNA amplification of PPV replicative form (RF), a 158-bp fragment was detected in agarose gels. This amplified fragment was shown to be specific for PPV DNA after Southern transfer and hybridization to a 20-base internal probe. The amplified fragment also contained a single EcoRI cleavage site. Various conditions, such as number of cycles and annealing temperature, were examined to optimize the conditions for detecting viral DNAs from infected cell cultures and swine fetal tissues. Four different isolates of PPV, NADL-8, NADL-2, KBSH and Kresse, and two other viruses, canine parvovirus (CPV) and pseudorabies virus (PRV), were included to determine specificity of amplification. Slot blot hybridization with a radiolabeled probe was used to evaluate the sensitivity of PCR amplification. The optimized protocol was specific for PPV detecting equally all four strains of PPV, but failing to amplify CPV or PRV sequences. The PCR method could detect at least 100 fg of viral replicative form (RF) DNA or the equivalent of 1 PFU of infectious virus. The applications of this method include routine detection of PPV in clinical samples and as a contaminant in mammalian cell lines.     

Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) method is a novel, sensitive and rapid technique which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for white spot syndrome virus (WSSV) detection was designed. A set of four primers, two outer and two inner primers, were designed from WSSV genome DNA. Reaction time and temperatures were optimized for 60 min at 65 degrees C, respectively. The detection limit (DL) using the LAMP method was up to 1 fg, when compared to 10 fg by nested polymerase chain reaction (PCR). Thus, standardized LAMP procedure was used to detect the presence of WSSV in the heart, stomach and lymphoid organ from infected shrimp. The study has developed a diagnostic procedure which is a rapid and highly sensitive for WSSV detection in shrimp.     

Diagnosis of equine gammaherpesvirus 2 and 5 infections by polymerase chain reaction

Summary Nested polymerase chain reaction (PCR) assays were developed for the detection of equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5) using the nucleotide sequences from the glycoprotein B (gB) gene of EHV2 and the thymidine kinase (TK) gene of EHV5. The simultaneous use of EHV2 specific and EHV5 specific primers in one nested amplification assay (multiplex PCR) enabled a rapid, specific and sensitive diagnosis for each virus. PCR was found to be 10³ times more sensitive than virus isolation by cell culture for EHV2 and 10⁶ for EHV5. In separate PCR assays, the routine detection limit after ethidium bromide staining was 0.6 fg for EHV2 plasmid DNA and 2.3 fg for EHV5 plasmid DNA, equivalent for both viruses to approximately 100 genome copies. The detection limits in multiplex PCR were 6 pg for EHV2 and 2.3 fg for EHV5, respectively. PCR assays were applied to studies of the epidemiology of EHV2 and EHV5 infections of racehorses and breeding mares in Victoria and New South Wales, Australia. Peripheral blood leukocytes from 31% of horses were positive for EHV2, 16% positive for EHV5, 8% positive for both viruses and 63% negative for both viruses. EHV2 PCR was also successfully used to detect EHV2 DNA in nasal secretions from horses. The multiplex PCR assay proved to be a rapid and reliable method for the simultaneous detection and differentiation of 2 related equine gammaherpesviruses.     

Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis.

In recent work, a species-specific Mycobacterium tuberculosis DNA fragment was cloned and sequenced. On the basis of its nucleotide sequence, two oligonucleotides were synthesized and used as primers for polymerase chain reaction (PCR) amplification. A 396-bp fragment was specifically amplified from the M. tuberculosis genome. No amplification was observed from any of 10 different mycobacterial strains, included those belonging to the M. tuberculosis complex. Neither was this fragment amplified from genomes of humans or different species of clinically important bacteria. The PCR product was detected by dot blot hybridization even when as little as 10 fg of purified M. tuberculosis DNA was used. This amplification method was subsequently used to detect and identify bacilli in different clinical samples, such as sputum, urine, and cerebrospinal fluid. A good correlation was observed between the results obtained with the PCR method that we describe and other diagnostic tests currently used. Thus, PCR amplification of this genomic fragment is proposed as a specific, rapid, and sensitive test for the diagnosis of infection with M. tuberculosis.     

Detection of White spot syndrome virus DNA in pond soil using a 2-step nested PCR

White spot syndrome virus (WSSV) continues to be the most pathogenic among the penaeid shrimp viruses. In this study, WSSV DNA was detected in pond soil samples using a 2-step nested PCR. Primers described previously were used for the first round of amplification and based on the sequenced amplicon, an inner primer was designed for the 2nd round of amplification. Using plasmid DNA (pET 100) containing the 211 bp target WSSV sequence, analytical sensitivity showed that the 2-step nested PCR protocol was able to detect down to 0.015 fg of the plasmid DNA, or approximately 2 copies of the target DNA sequence. Persistence of WSSV DNA in pond soil samples after various time intervals was determined. WSSV-specific PCR product (161 bp) was still present in the soil samples even after 10 months of storage. The effect of soil heat treatment on the WSSV DNA was also examined. Soils were subjected to 25, 37, 50 and 70 degrees C for 1, 3 and 5 days. The results showed that PCR amplifiable WSSV DNA was still present even after 5 days at 70 degrees C. To our knowledge, this is the first report on the detection of WSSV DNA in soil samples. Based on these findings, it is concluded that the persistence of viral DNA in soil habitats may be an important aspect of WSSV ecology and may have an implication for viral transmissibility.     

Detection of H-1 parvovirus and Kilham rat virus by PCR.

H-1 virus and Kilham rat virus (KRV) are autonomous parvoviruses which generally cause subclinical infections in rats and can cause persistent infections in cell cultures. In this study, primer sets specific for either H-1 or KRV were designed on the basis of DNA sequence comparisons of the rodent parvoviruses. The specificities of the H-1 and KRV-specific primer sets were determined by testing viral preparations of seven different parvoviruses and nine other viruses known to infect rodents. The H-1-specific PCR assay amplified the expected 254-bp product only in the presence of H-1 viral DNA and was able to detect as little as 100 fg of H-1 viral DNA. The KRV-specific PCR assay generated the expected 281-bp product only when KRV viral DNA was used as the template and was able to detect as little as 10 pg of KRV viral DNA. Each assay was able to detect its respective virus in tissues from rats experimentally infected with H-1 or KRV. In contrast, no product was amplified by either assay with tissues from mock-infected rats. Our findings indicate that these PCR assays provide rapid, specific, and sensitive methods for the detection of H-1 or KRV infection in rats and cell culture systems.     

Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens

We describe a novel adaptation of the TaqMan PCR assay which potentially allows for highly sensitive detection of any eubacterial species with simultaneous species identification. Our system relies on a unique multiprobe design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair and is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step effectively decontaminates or removes background DNA. The TaqMan system described reliabAly detected 14 common bacterial species with a detection limit of 50 fg. Further, highly sensitive and specific pathogen detection was demonstrated with a prototype species-specific probe designed to detect Staphylococcus aureus. This assay has broad potential in the clinical arena for rapid and specific diagnosis of infectious diseases.     

Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.     

Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon

A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794 bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277 bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in approximately 10 fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406 bp) or YHV-specific (277 bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.     

Molecular characterization and differentiation of opisthorchiid trematodes of the species<Emphasis Type="Italic"> Opisthorchis felineus</Emphasis> (Rivolta, 1884) and<Emphasis Type="Italic"> Metorchis bilis</Emphasis> (Braun, 1790) using polymerase chain reaction

Adult specimens of the opisthorchiid liver fluke species Opisthorchis felineus and Metorchis bilis could be identified for the first time by molecular biological methods using species specific primers (OF and MB primers) in the polymerase chain reaction (PCR). The OF or MB primers were based on a part of the mitochondrial cytochrome c oxidase I gene. A specific product of approximately 200 bp could be amplified for O. felineus by means of the specific O. felineus primers. By contrast, the amplification of M. bilis DNA with MB primers produced a fragment of approximately 110 bp. A specificity of 100% could be demonstrated for both primer pairs. The sensitivities of the PCRs were 10 pg for the O. felineus DNA and 100 fg for the M. bilis DNA.