Diagnosis of acute and latent varicella-zoster virus infections using the polymerase chain reaction.

A simplified assay for the diagnosis of varicella-zoster virus (VZV) infections based on the polymerase chain reaction (PCR) is described. Omitting the procedures for extraction and purification of DNA, the crude vesicle fluid materials were used for PCR. Moreover, hybridization was not necessary for detection of the amplification products because they were already visible after ethidium bromide staining of the electrophoresis gel. Results could therefore be obtained within one day. In comparison to virus isolation, PCR proved much more rapid, highly sensitive, and specific. DNA extraction and a double PCR assay with nested primers were necessary for detection of latent VZV infections in trigeminal and thoracic ganglia. The data suggest that the procedures described are universally applicable to several types of specimens dependent on the calculated amount of target DNA.     

The detection of beet western yellows virus and beet mild yellowing virus in crop plants using the polymerase chain reaction.

Oligonucleotide primers were synthesised corresponding to conserved sequences between three isolates of beet western yellows virus (BWYV), flanking a 913 base fragment of BWYV genomic RNA. Using the polymerase chain reaction (PCR), these primers successfully amplified the target fragment in total RNA extracts from two oilseed rape plants infected with different isolates of BWYV. The PCR products were readily detected by staining with ethidium bromide following agarose gel electrophoresis, but the limit of detection could be increased further by Southern blotting. However, three isolates of beet mild yellowing virus (BMYV) in sugar beet did not give a signal which could be detected by ethidium bromide staining, although the target fragment could be detected by Southern blotting. The primers used have the potential to detect BWYV in crops with far greater sensitivity than enzyme-linked immunosorbent assay or nucleic acid hybridisation (dot-blotting) and may be capable of distinguishing between BWYV and BMYV. The application of PCR to detection and distinction of luteoviruses in general is discussed.     

Separation and detection of DNA polynucleotides using capillary electrophoresis. Application to detection of polymerase chain reaction-amplified human immunodeficiency virus and HLA DNA.

The polymerase chain reaction (PCR) is a technique for rapid amplification of target DNA sequences. During the past several years, a large number of research applications of PCR have appeared, many of which may prove to be useful clinically. We report the use of capillary electrophoresis, a fully automated technique, as an alternative to polyacrylamide gel electrophoresis for the detection of PCR-amplified viral and cellular DNA. We describe conditions for rapid separation, detection, and discrimination of PCR products from the human immunodeficiency virus type 1 gag gene and the HLA-DQ-alpha gene amplified from the human immunodeficiency virus provirus-containing U1.1 cell line. The sensitivity achieved with the use of capillary electrophoresis analysis was roughly equivalent to that of ethidium bromide staining of polyacrylamide gel electrophoresis gels. Further refinement of capillary electrophoresis for automated detection and quantitation of PCR-amplified products should expedite more widespread application of PCR analysis in the clinical laboratory.     

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence.

We have developed a sensitive and quantitative assay for hepatitis B virus (HBV) DNA in serum or plasma in which PCR and then microtiter hybridization analysis are used. Assay of HBV DNA in serum or plasma is important for demonstrating viral replication, indicating and monitoring antiviral therapy, determining the infectivities of virus carriers, and ensuring the safety of blood products. Under optimum conditions PCR can amplify one HBV DNA molecule to 10(8) copies, but detection of this amount of DNA still requires hybridization with labelled probes or a nested PCR. We labelled one strand of the PCR product with a biotinylated primer. The double-stranded amplicon was incubated in streptavidin-coated microplate wells. The nonlabelled strand was removed after denaturation of the double-stranded DNA with alkali, and the bound strand was hybridized with a peroxidase-coupled single-stranded probe. The amount of bound peroxidase was measured in a luminometer. Four picograms of amplicon was detectable in this system, whereas conventional ethidium bromide staining requires a 1,000 times higher amplicon concentration. The performance of the new assay was compared with those of nested PCR and a PCR system that uses a digoxigenin label, hybridization to a solid-phase adsorbed probe, and colorimetric detection. The chemiluminescence assay was found to be almost as sensitive as nested PCR and approximately five times more sensitive than the colorimetric test.     

Optimized PCR assay for the detection of TT virus

A polymerase chain reaction (PCR)-based procedure for the detection of TT virus DNA is described. In this method. total nucleic acid extracted from a small volume of serum or plasma is utilized as a template in PCR employing TT virus specific primers designed to highly conserved regions of the virus genome. Additional sensitivity is obtained by carrying out a second round of amplification. Reactions are analyzed by agarose gel electrophoresis, and samples having an ethidium bromide stainable fragment of the appropriate size in the first and/or second amplification are designated as positive. This protocol allows for the rapid and sensitive detection of TT virus in human plasma or serum.     

Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique.

We have developed a rapid procedure for the detection of serum hepatitis B virus (HBV) DNA using the polymerase chain reaction (PCR) technique. HBV DNA is released from virions by incubating serum with 0.1 M NaOH for 60 min at 37 degrees C. The mixture is brought to neutral pH with HCl, and the HBV DNA sequences are detected by agarose gel electrophoresis and ethidium bromide staining after PCR amplification with two successive sets of primer pairs. The detection limit of this method (i.e., 10(-5) pg of HBV DNA) is equivalent to that previously determined by one round of PCR amplification and Southern blot hybridization analysis. The advantages are that the assay can be completed in 1 day, is very sensitive, and does not require the use of radiolabeled reagents.     

The detection of bovine leukemia virus proviral DNA by PCR-ELISA.

A sensitive non-radioactive microplate hybridization assay for the detection of proviral DNA of bovine leukemia virus (BLV)-specific polymerase chain reaction (PCR) product is described. The PCR products are labeled by adding digoxigenin-dUTP to the nested PCR reaction and are captured by a microtitre plate coated with oligonucleotide probe, which is complementary to the inner region of the amplification product. Captured products are reacted with an anti-DIG Fab fragment conjugated to peroxidase, and detected using a colorimetric reaction. The PCR-enzyme linked immunosorbent assay (ELISA), detecting as low as 10(-4) ng of proviral DNA in a background of 1 microg of BLV-negative DNA, was up to 100-fold more sensitive than ethidium bromide staining, and showed equal sensitivity to Southern blot hybridization. Using this method it was possible to monitor the presence of proviral DNA in four sheep infected experimentally with BLV, over a 10 months postinfection period, as well as in 29 cattle infected naturally. The test is rapid and highly sensitive and is a useful additional tool for the detection of BLV-infected animals.     

干血斑滤纸片中人免疫缺陷病毒1型前病毒基因的检测

目的建立套式引物聚合酶链反应（ＰＣＲ），用于检测滤纸干血斑中的人免疫缺陷病毒１型（ＨＩＶ１）前病毒ｐｏｌ基因ＤＮＡ片段。方法采集ＨＩＶ１感染者的全血约５０μｌ滴在经ＥＤＴＡ－蛋白酶Ｋ预处理并干燥的滤纸片上，室温下干燥，将滤纸片密封于塑料袋中，在室温及４℃下保存１～６４周后，分别将滤纸片置０５ｍｌ试管中直接进行ＨＩＶ１前病毒ｐｏｌ基因的外侧引物ＰＣＲ检测，然后进行内侧引物的ＰＣＲ检测。结果经ＥＤＴＡ－蛋白酶Ｋ预处理的滤纸片在４℃下保存４０周、在室温下保存２４周仍可检出ＨＩＶ１前病毒目的基因。根据ＰＣＲ产物的琼脂糖凝胶电泳溴化乙锭染色带形并参比实验设立的标准对照可直接判断结果。结论该方法具有快速、特异、敏感的特点，敏感性可以达到检出１０个靶ＤＮＡ分子。样品采集后可通过邮件传递至中心实验室，特别适合于ＨＩＶ１感染的确证及筛检     

Simple, sensitive, and specific detection of human immunodeficiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers.

A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for the detection of human immunodeficiency virus type 1 (HIV-1) is described. We have improved all three PCR steps: sample preparation, DNA amplification, and detection of the amplified product. Some of the improvements have been described previously, but they have never been combined into a complete PCR protocol. Peripheral blood mononuclear cells were lysed directly in a buffer containing sodium dodecyl sulfate, Triton X-100, and proteinase K. This crude cell lysate was amplified in a two-step PCR, first with outer primers and then with inner primers nested within the first primers. The PCR product was visualized by agarose gel electrophoresis and ethidium bromide staining. Thus, we avoided conventional DNA extraction as well as hybridization for the detection of the PCR product. The samples were analyzed with four sets of nested primers (JA4 through JA7, JA9 through JA12, JA13 through JA16, and JA17 through JA20) designed to amplify HIV-1 gag, env gp120, env gp41, and pol sequences, respectively. We were able to amplify HIV-1 sequences in all samples from 90 HIV-1-seropositive individuals with mostly mild symptoms. Of these individuals, 24 were negative in HIV-1 isolation and 9 were selected because they were infected by African and Haitian HIV-1 strains. Eighty-five (94%) individuals were positive with at least three of four primer sets. Samples from 26 healthy blood donors, as well as cells infected in vitro with human immunodeficiency virus type 2 and human T-cell leukemia virus type I, were negative in PCR, thus demonstrating the specificity of the amplification.     

Comparison of white spot syndrome virus PCR-detection methods that use electrophoresis or antibody-mediated lateral flow chromatographic strips to visualize PCR amplicons

White spot syndrome virus (WSSV) PCR-detection methods that used electrophoresis or lateral flow chromatographic strips (LFCS) were to compare and visualize PCR amplicons. Real-time PCR was used to prepare a stock template solution containing 2.85 x 10 (6) copies WSSV/microl from WSSV-infected shrimp. Serial stock dilutions were used as templates for PCR amplification of a WSSV-specific DNA fragment that was detected either in ethidium bromide stained agarose electrophoresis gels or on a chromatographic strip where it interacted with antibody to markers labeled on hybridization complex. PCR amplification employed both 1-step PCR and semi-nested PCR methods. By using 1-step PCR, the LFCS method (100 copies) gave 10 times higher sensitivity than gel electrophoresis (10(3) copies). A combination of a semi-nested PCR with LFCS gave a comparable sensitivity to those with commercial kits for nested PCR (20 copies). In addition, LFCS confirmed amplicon identity, avoided handling of carcinogenic ethidium bromide and could be completed in approximately 20-30 min post-PCR compared with 1h for gel electrophoresis. The costs for the two methods were comparable. In conclusion, semi-nested PCR followed by LFCS is a safe and rapid alternative method for detection of WSSV that provides sensitivity similar to that obtained by standard nested PCR methods.     

New method for plague surveillance using polymerase chain reaction to detect Yersinia pestis in fleas.

Yersinia pestis, the plague bacillus, infects a variety of mammals throughout the world and is transmitted by fleas. We developed a polymerase chain reaction (PCR) test using primers designed from the Y. pestis plasminogen activator gene to directly detect plague-infected fleas. As few as 10 Y. pestis cells were detected, even in the presence of flea tissue, by PCR and then agarose gel electrophoresis and ethidium bromide staining. The feasibility of the assay was demonstrated by using naturally infected Xenopsylla cheopis fleas. The detection of Y. pestis in fleas by PCR provides a rapid and sensitive way to monitor plaque in wild animal populations, allowing public health officials to better assess the potential risk of transmission to humans.