End-labeled Probes Research Articles

Extraction and quantitation of cytokine mRNA from human epidermal blister roofs.

The demonstration of cytokine mRNA expression in epidermal cells by the polymerase chain reaction technique preceded by reverse transcription (RT-PCR) requires linear test conditions (i.e. that the product obtained after amplification reflects the relative amounts of starting material) and high reproducibility, specificity, and sensitivity. By combining well-defined techniques for mRNA extraction and concentration measurement with a sensitive and well-calibrated RT-PCR technique, we demonstrated the presence of IL-1 alpha, IL-1 beta, IL-6, and TNF alpha in epidermal cells obtained from suction blister roofs from normal volunteers. Messenger RNA was extracted with superparamagnetic oligo(dT)25 Dynabeads, and the amount of mRNA was measured by spectrophotometry using a Beckman 5-microliters Ultra-Microcell prior to RT-PCR. Linear PCR conditions were obtained by carefully titrating the amounts of mRNA and the number of cycles. Reproducibility was estimated at different steps of the procedure, and the specificity of the enhanced cDNA products was verified by liquid hybridization with end-labelled probes. We suggest that this combination of techniques might prove useful for the simultaneous assessment of the expression of various cytokines from small samples of fresh human epidermal cells.

Efficient synthesis of 32P-labeled single-stranded DNA probes using linear PCR; application of the method for analysis of strand-specific DNA repair

We have developed a rapid method to synthesize radioactively labeled single-stranded DNA probes suitable for strand-specific analysis of single copy genes on Southern blot. Linear PCR with 10 μCi α32P-aATP (3000 Ci/mmol) as the only dATP source enabled us to generate strand-specific DNA probes with high specific activity. The probes synthesized by this method have higher specific activities and the same strand specificity compared to the end-labeled single-stranded DNA probes obtained from single-stranded M13mp18/19 vectors. Application of the method for strand-specific analysis of ultraviolet-induced DNA lesions in defined DNA sequences significantly improved the hybridization signal.

Single-tube, noninterrupted reverse transcription-PCR for detection of infectious bursal disease virus.

An assay protocol based on single-tube, noninterrupted reverse transcription-PCR (RT-PCR) for the detection of infectious bursal disease virus (IBDV) is described. After the conditions for RT-PCR had been optimized, a primer set framing a region within the gene coding for IBDV VP2 protein was used to amplify a 318-bp fragment of the IBDV genome. Amplified product was detected with three strains of IBDV, whereas none was obtained from uninfected bursal tissue or seven unrelated avian viruses. The sensitivity of this RT-PCR was tested with purified viral RNA from three strains of IBDV. The detection limit was 10 fg in an ethidium bromide-stained gel. In addition, this assay system was used to detect IBDV in bursal-tissue specimens from commercially reared chickens. The identity of the amplified products from the tissue specimen preparation was determined by using a rapid, simple procedure in which internally nested, end-labeled probes were used.

A simplified method for PCR detection of hepatitis C virus RNA from human serum.

1The Laboratory of Virology and Parasitology, The Lindsley F. Kimball Research Institute of the New York Blood Center, New York, New York 10021; 2Roche Molecular Systems, Branchburg, New Jersey 08876-1760 Hepatitis C virus (HCV) is responsible for most cases of post-transfusion non-A, non-B hepatitis. (1~ PCR is a highly sensitive test for detection of HCV RNA in biological specimens and is a potentially powerful tool in laboratory diagnosis of HCV infection. Serum HCV RNA determinat ion may eventually be required for prevention of blood-borne HCV transmission. ~2~ To control contaminat ion this would require automation of the PCR technology, a development that is presently hampered by technical difficulties. One of the main obstacles is the necessity of extracting viral RNA before amplification, a t ime-consuming step that is not readily automatable. Several alternative methods for sample processing for HCV reverse transcription (RT)PCR have been reported, ~3'4~ all tending toward a much shorter and simpler procedure. Here, we describe a simple method for RT-PCR amplification of HCV RNA from h u m a n serum that does not require any extra RNA extraction step. The method is based on the f inding that Triton X-100 can release HCV RNA from the viral envelope and that this can be efficiently reverse-transcribed from the crude lysate. The protocol is as follows: One to two microliters of serum diluted with 8-9 i~l of fetal calf serum (HyClone) is added directly to the RT mix (50 mM KCI, 10 mM Tris-HCl at pH 9.0, 4 mM MgC12, 0.1% Triton X-100, 20 units of RNasin ribonuclease inhibi tor (Promega Corp., Madison, WI), 150 ng of an HCVspecific antisense primer 20 (5'-GCT CAT GGT GCA CGG TCT A, position 13 to + 6 of the viral genome), 200 I~M dNTPs, and 7.5 units of avian myeloblastosis virus (AMV)-RT (Promega) in a final volume of 20 i~l. Fetal calf serum is used as a stabilizer to prevent coagulation during the denaturation step following cDNA synthesis. In view of the known homology between HCV and animal pestiviruses, ~s~ and the possibility of contaminat ion of fetal calf serum by bovine viral diarrhea virus (BVDV), ~6~ care should be taken in both the design of PCR primers and in the use of virusscreened fetal calf serum. BVDVscreened fetal calf serum is widely available. New lots of fetal calf serum are evaluated for their ability to yield expected titer of our positive control HCVinfected serum and for negative results with a panel of noninfected control sera. cDNA synthesis is carried out at 43~ for 60 min followed by denaturat ion for 15 min at 90~ PCR mix is added [50 mM KC1, 100 mM Tris-HC1 at pH 9.0, 2 mM MgC12, 0.1% Triton X-100, 2.5 units of Taq DNA polymerase (Promega), 200 IM 22, 5'-ACT CGC AAG CAC CCT ATC A (nucleotides 293-312)] located in the 5' nonstructural region of the viral genome, generating a PCR product of 267 bp. PCR cycles are as follows: The first five cycles (94~ for 1.5 min, 72~ for 5 rain) were followed by 35 cycles of 94~ for 1.5 min, 60~ for 2 min, and 72~ for 3 min, followed by 72~ for 7 min. Amplified products were detected by oligomer hybridizat ion (7~ with a 32p. end-labeled probe (positions 161-180; 5'-GAG TAC ACC GGA ATT GCC AG) located between the primers. The probetarget duplex was separated from unhybridized probe by NuSieve 3:1 agarose gel electrophoresis. The gel was then dried and autoradiographed. Alternatively, PCR, using a biotinylated antisense primer, was followed by detection of PCR product using an experimental enzyme-l inked hybridization capture assay kindly provided by Roche Molecular Systems (Branchburg, NJ). The present method detected HCV RNA using 1 i~l of 10s to 107 dilutions of our positive control serum that titered 10-s/50 I~l by nested PCR after a standard guanid in ium/phenol /ch loroform extraction and alcohol precipitation. (2~ This control serum was chosen as having the highest titer among 19 PCR-positive blood donors sera tested. (2~ Thus, the present method is 50-500 times more sensitive than nested PCR. This was confirmed by analysis of five different plasma specimens that had been titrated by RT-PCR after guan id in ium/pheno l / chloroform extraction. These sera had been chosen to have intermediate titer (10 -2) in nested PCR titrations. As shown in Table 1, the direct method was 10to lO0-fold more sensitive for dilution of HCV RNA in ~'s sera even when the volume of sample tested is not taken into account. In one serum the two methods were of equivalent sensitivity. The specificity of the method was demonstrated by our experience in

The detection and quantification of feline immunodeficiency provirus in peripheral blood mononuclear cells using the polymerase chain reaction

The polymerase chain reaction method (PCR) was used to detect feline immunodeficiency virus proviral DNA in peripheral blood mononuclear cells (PBMC) of a group of 8 experimentally infected cats. The proportion of PBMC containing provirus was determined from 6 to 32 weeks post inoculation (p.i.) by performing PCR on serially diluted samples of PBMC. Primers from the p15 and p24 regions of the gag gene were used and Southern hybridization using an end-labelled probe was required to confirm primer-specific products. Provirus was detected in 5 of 8 cats by 6 weeks p.i. in 50000 PBMC, and in all 8 infected cats by 8 weeks p.i. Provirus was not detected in PBMC from any of 3 FIV negative cats. The proportion of PBMC containing provirus in individual cats ranged from 1 in 70 to 1 in 99600 PBMC. There was no significant decline over time in the proportion of PBMC containing provirus. Sequencing of a segment (287 bases) of the gag region of a West Australian FIV isolate (T90) revealed only slight nucleotide divergence from the North American Petaluma and PPR isolates and wider divergence from the Japanese TM2 clone.

Chromosomal Mapping of the Human CNP Gene Using a Meiotic Crossover DNA Panel, PCR, and Allele-Specific Probes

The human 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) gene is located on chromosome 17, as determined by PCR of somatic cell hybrid DNA panels and confirmed using a mouse-human hybrid containing only human chromosome 17. A polymorphic site (C, T) was previously described at nucleotide 1215 within the most 3' intron of the gene. Nested PCR primer pairs were designed to amplify across this site, and PCR products were hybridized to end-labeled allele-specific probes. To localize further the CNP gene within chromosome 17, a two-step strategy was used. First, dot blots containing DNA from the parents of 10 three-generation families were screened to identify the potentially informative families. Second, 53 members of four selected families were typed at this locus. Previous studies had shown that the 29 siblings present in these four families carry a total of 84 meiotic breakpoints on chromosome 17. Based on the genotypes observed in these 29 siblings, the human CNP gene was localized to a fragment on 17q bounded by THRA1 (thyroid receptor A1) and NGFR (nerve growth factor receptor), a genetic distance of approximately 6 cM.

Primer extension analysis provides a sensitive tool for the identification of PCR-amplified DNA from HIV-1

Primer extension analysis was evaluated as a means to identify PCR-amplified DNA from HIV-1. Solution hybridization with radioactive labeled oligonucleotides followed by an extension reaction with Klenow fragment of Escherichia coli DNA polymerase I and subsequent separation on denaturing polyacrylamide gels reveals single stranded DNA products of the predicted size. The specificity of the reaction is further demonstrated by specific endonuclease digestion. The analysis is more sensitive than Southern blotting and about equally sensitive as Slot blot analysis when double-stranded DNA probes are used for hybridization. With end-labeled oligonucleotide probes, primer extension analysis proved an order of magnitude more sensitive than membrane hybridization. The analysis also allows quantitation of amplified DNA from 1 pg to about 1 ng of DNA product. Under the conditions described for amplification, primer extension analysis is capable of detecting a single HIV-1 plasmid DNA molecule in the presence of 1 μg of total DNA. 3'-end mismatching of the oligonucleotide probe does not result in a significantly altered detection limit. Primer extension analysis can also be carried out with at least three different PCR-amplified DNAs in the same reaction tube.

DNA typing with digoxigenin-11-dideoxyuridinetriphosphate-labelled oligonucleotide probes enables non-radioactive analysis using a dual detection system: application for screening HLA-DQA1 polymorphisms.

We describe a standardized, highly sensitive, nonradioactive detection procedure for HLA class-II typing of DQA1 alleles and suballeles which has important and cost-effective application in studying HLA disease associations. The procedure involves polymerase chain reaction-based target DNA sequence amplification and dot blotting followed by stringent hybridization with digoxigenin-11-dideoxyuridine triphosphate 3'-end-labelled allele-specific oligonucleotide probes. The dual detection system described here makes this procedure very versatile.

Geographical clusters of dengue virus type 2 isolates based on analysis of infected cell RNA by the chemical cleavage at mismatch method

Genetic variation in 12 strains of dengue virus type 2, isolated from several epidemic areas in different years, was studied by chemical cleavage at mismatched cytosine in DNA:RNA heteroduplexes. End-labelled cDNA probes derived from the E and NS2A genes of the New Guinea C strain were hybridized to total RNA extracted from cells infected by individual isolates. Following modification of mismatched cytosine by hydroxylamine and nucleic acid strand cleavage by piperidine, the resulting fragments of radiolabelled probe were analysed by electrophoresis and autoradiography. The patterns of bands generated corresponded to the geographical groupings of the isolates. Thus this method is suitable in epidemiological studies for rapidly surveying a large number of isolates for genetic variation in a particular gene of interest.

Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements.

A20 was first identified as a tumor necrosis factor (TNF) primary response transcript encoding a 790-amino acid protein with a unique zinc finger motif. Recently, A20 was shown to protect cells from TNF-induced cytotoxicity in a variety of cell lines. Nuclear run-on studies previously established that TNF activates A20 at the transcriptional level. To further characterize the mechanism by which TNF activates the A20 gene, we have cloned the A20 5'-flanking sequences and identified TNF-responsive elements within the promoter. The transcription initiation site was mapped by both primer extension and S1 nuclease protection experiments to a position 4.2 kilobases (kb) upstream of the initiator methionine; the first and second exon were separated by a 3.9-kb intron. Sequences upstream of the transcription start site were 76% GC-rich and contained six Sp1 binding sites and a TATA-like sequence at -29 but lacked a consensus CCAAT site. Transfection of Jurkat T-cells with an array of A20 promoter CAT constructs showed that two kappa B elements residing at -54 and -66 were required for induction by TNF. Supporting this notion, DNA electrophoretic mobility shift assays using nuclear extracts from unstimulated and TNF-stimulated Jurkat cells demonstrated kappa B-specific binding of a TNF-activated factor to an end-labeled probe containing the two A20 kappa B sequences. Finally, evidence obtained from cotransfection experiments showed that A20 negatively regulated its own expression.

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.

An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts.

A posttranscriptional modification (C-to-U) at specific positions of plant mitochondrial mRNA leads to changes in the amino acid sequence as well as to the emergence of novel initiation or termination sites. This phenomenon, named RNA editing, has been described for several mitochondrial genes from different plant sources. We have found recently that RNA editing of the ATP synthase subunit 9 (atp9) mRNA involves eight changes including the creation of a new stop codon. In this article, we describe an in vitro system devised to follow the editing of wheat mitochondrial atp9 mRNA. Nonedited mRNA was obtained to serve as substrate for this reaction by in vitro transcription of the corresponding gene with T7 RNA polymerase. The source of conversion factor(s) was a soluble fraction obtained from purified wheat mitochondria lysed with salt and detergent. Edited RNA molecules were detected by hybridization with an end-labeled synthetic oligodeoxynucleotide probe complementary to a short region containing four editing events. Optimal conditions for the in vitro RNA editing reaction were determined. The reaction is sensitive to high temperature and protease digestion. Pretreatment with micrococcal nuclease decreased RNA editing activity in the mitochondrial extract, suggesting that a nucleic acid is necessary for the enzymatic reactions. Analysis of the edited mRNA showed that the in vitro reaction led to the same products as those observed in vivo.

Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides

Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5'-end or enzymically at the 3'-end. Following specific in situ hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti-DIG Fab fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody-enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gram-negative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence.

Co-amplification of specific sequences of HCV and HIV-1 genomes by using the polymerase chain reaction assay: a potential tool for the simultaneous detection of HCV and HIV-1

A rapid and simple method using the polymerase chain reaction (PCR) was devised for the co-amplification and simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) specific sequences in the same serum sample. Genomic RNA was extracted from 13 blood donor sera that were reactive in ELISA for both anti-HCV and anti-HIV-1. The extracted RNA was reverse transcribed into cDNA and amplified using nested primer pairs (SN01 and SN04; SN02 and SN03) based on the HCV prototype sequence of clones 37b and 81, and SK 38 39 for HIV-1 simultaneously. PCR products were analyzed by liquid hybridization or Southern blot hybridization with 32P end-labeled oligonucleotide probes from the regions between the primer pairs, excluding the primer sequences. HCV-RNA was detected in all 13 (100%) samples tested; HIV-RNA was detected in 11 (85%) samples. The ability to co-amplify specific sequences from two different viral genomes in the same reaction mixture offers the possibility of simultaneous detection and diagnosis of more than one viral agent in serum samples of infected individuals.

Characterization of pearl millet mitochondrial DNA fragments rearranged by reversion from cytoplasmic male sterility to fertility

Cloned pearl millet [Pennisetum glaucum (L.) R. Br.] mitochondrial (mt) DNA fragments rearranged by spontaneous reversion from cytoplasmic male sterility (cms) to fertility were characterized by restriction mapping, hybridization with maize mt genes, and transcription analyses. The clones characterized were a 4.7-kb fragment found only in the male-sterile cytoplasm and lost upon reversion to fertility, a 10.9-kb fragment found in all cytoplasms and not changed by reversion, a 13.6-kb fragment found in the male-sterile and -fertile normal cytoplasms and lost in seven of the eight revertants studied, and a 9.7-kb fragment not found in the male-sterile cytoplasm but produced by reversion from male sterility to fertility. The restriction maps verified that the four cloned pearl millet fragments contained two sets of repeated sequences, one on the 4.7-, 10.9-, and 13.6-kb fragments, the other on the 13.6- and 9.7-kb fragments. The rrn18, rrn5, and coxI genes were located in the repeated regions of the 4.7-, 10.9-, and 13.6-kb cloned fragments. The correlation of reversion (eight independent events) with the loss of fragments containing the rrn18, rrn5, and coxI genes suggests that those lost fragments and their gene content could be responsible for the expression of cms. Transcriptional analyses using both Northern blots and end-labeled mtRNA probes verified that transcripts homologous to the rrn18 and coxI genes were present in pearl millet total mtRNA. However, no transcript differences were detected among cms, revertant, and fertile normal cytoplasms, suggesting that the reversion process involves mutational changes that may not affect transcript size. Transcript analyses indicated that the 10.9-kb clone contained an unidentified gene on the end opposite the rrn18 gene; however, since it was present in all cytoplasms, it is not believed to be involved in cms.

DNA sequence analysis of revertants of the hisD3052 allele of Salmonella typhimurium TA98 using the polymerase chain reaction and direct sequencing: Application to 1-nitropyrene-induced revertants

We have used the polymerase chain reaction (PCR) to speed the DNA sequence analysis of revertants of Salmonella typhimurium TA98. Briefly, a crude DNA extract from a single colony was prepared and used in an asymmetric PCR to amplify a 328-bp fragment containing the hisD3052 mutation approximately in the center. Following ultrafiltration, the ssDNA was sequenced using an end-labeled probe and dideoxy sequencing. The most frequent mutation among the revertants was a −2 deletion of GC or CG within the sequence CGCGCGCG, which is upstream of the hisD3052 mutation. This deletion occurred in 38% ( 6 16 ) of the spontaneous (−S9) revertants and in 94% ( 15 16 ) of a set of 1-nitropyrene-induced revertants. Other mutations, mostly deletions but also some complex mutations (i.e., single mutational events involving a combination of insertions, deletions, and substitutions), occurred within quasipalindromic regions of DNA. Possible mutational mechanisms are discussed, and the results with 1-NP are compared to those obtained in other systems.

Presence of hpv 16 sequences in laryngeal carcinomas

Human papillomavirus types HPV 16 and HPV 11 DNA sequences were analyzed in normal and neoplastic tissues of the larynx, using the technique of polymerase chain reaction (PCR). An amplified region of E6 ORF was hybridized with 3' end-labelled oligonucleotide probe. Twenty six out of 48 (54%) squamous-cell carcinomas, and 3 out of 3 verrucous-cell carcinomas hybridized with HPV 16 DNA sequences, whereas we did not detect HPV 11 sequences. HPV 16 DNA sequences were also found in normal, autologous mucosa and lymphnode metastases, although these were absent in other tissues analyzed. HPV-16-positive tumors were most frequently poorly differentiated squamous-cell carcinomas.

Detection of infectious pancreatic necrosis virus (IPNV) RNA by hybridization with an oligonucleotide DNA probe

Marine farming of Atlantic salmon ( Salmo salar) is growing rapidly in Norway, and fish diseases have now become one of the biggest obstacles for this new industry. Infectious pancreatic necrosis virus (IPNV) is commonly found in fish from Norwegian sea farms. Diagnosis of IPNV infection is, at present, mainly based on virus isolation in cell cultures and identification by neutralization tests (NT). A DNA-RNA hybridization assay was developed using a 24 base DNA oligonucleotide probe. This is homologous to a part of the nucleotide sequence of the IPNV genome coding for a protease. RNA extracted from IPNV and harvest from infected cell cultures were fixed to nylon filters and hybridized with the 32P end-labelled probe. The results showed that the probe specifically identified IPNV from these two materials, both for the three different virus strains (Ab, Sp and VR-299) used, and for several different field isolates. It did not hybridize with reoviruses or non-infected cell cultures used as controls. These results indicate that the probe is not serotype specific, and furthermore that RNA extraction is not required before hybridization. This method may be a useful alternative to NT for routine identification of IPNV, particularly when non-radioactive labelling of the probe is introduced.

Majority of RNA which co-purifies with unactivated rat hepatic glucocorticoid-receptor complexes is nonspecific and not receptor-associated

Molybdate-stabilized, unactivated rat hepatic glucocorticoid-receptor complexes were purified by a three-step procedure which includes affinity chromatography, gel filtration and anion exchange chromatography. Following elution of unactivated steroid-receptor complexes from the final DEAE-cellulose column, RNA which remained bound to the anion exchange resin was eluted with l M KCl. This RNA was small and heterogeneous in size. Equivalent amounts of RNA were detected after a mock purification which was devoid of receptors, suggesting that the presence of this RNA is not dependent on that of receptors. Both a [ 32P]DNA complementary to the RNA eluted from DEAE-cellulose and a [ 32P]DNA probe synthesized from total rat liver RNA gave similar results when hybridized to total rat liver RNA. These data indicated that the RNA which co-purified with unactivated receptors through the first two steps was very similar to total RNA in overall composition. Virtually identical hybridization patterns were also detected when end-labeled probes generated from the DEAE-cellulose eluted RNA or total liver RNA were hybridized to total genomic rat DNA, suggesting that the RNA eluted from the anion exchange resin is not specific or unique. Although these results do not exclude the possibility that there could be specific RNA species associated with the unactivated glucocorticoid receptor, they do indicate that the majority of the RNA eluted from DEAE-cellulose following elution of receptor complexes appears indistinguishable from total rat liver RNA and can be detected in parallel mock purifications.

Enhanced detection of a plant pathogenic mycoplasma-like organism by polymerase chain reaction.

According to the sequence of cloned genomic DNA of clover proliferation (CP) MLO, two polymerase chain reaction (PCR) primer pairs were synthesized to specifically amplify two CP MLO DNA fragments from crude nucleic acids containing CP MLO DNA and host plant DNA. The first primer pair guided the amplification of a 196-bp DNA fragment of CP MLO. The second primer pair directed the amplification of a 109-bp DNA fragment of CP MLO. PCR products were identified by liquid hybridization using 5' end-labeled sequence-specific internal probe followed by 8% polyacrylamide gel electrophoresis and direct sequencing of PCR products. A minimum of 2.5ng nucleic acids were needed to detect CP MLO when PCR was not applied, whereas a minimum of only 2.5×10-5ng nucleic acids were needed when the 109-bp CP MLO DNA fragment was amplified by PCR, and a minimum of 2.5×10-8ng nucleic acids were needed to detect CP MLO when the 196-bp CP MLO DNA fragment was amplified through PCR. No DNA fragments were amplified when nucleic acids from healthy periwinkle plants were used as PCR templates.