Proposal of an Appropriate Decalcification Method of Bone Marrow Biopsy Specimens in the Era of Expanding Genetic Molecular Study

BACKGROUND: The conventional method for decalcification of bone specimens uses hydrochloric acid (HCl) and is notorious for damaging cellular RNA, DNA, and proteins, thus complicating molecular and immunohistochemical analyses. A method that can effectively decalcify while preserving genetic material is necessary. METHODS: Pairs of bilateral bone marrow biopsies sampled from 53 patients were decalcified according to protocols of two comparison groups: EDTA versus HCl and RDO GOLD (RDO) versus HCl. Pairs of right and left bone marrow biopsy samples harvested from 28 cases were allocated into the EDTA versus HCl comparison group, and 25 cases to the RDO versus HCl comparison group. The decalcification protocols were compared with regards to histomorphology, immunohistochemistry, and molecular analysis. For molecular analysis, we randomly selected 5 cases from the EDTA versus HCl and RDO versus HCl groups. RESULTS: The decalcification time for appropriate histomorphologic analysis was the longest in the EDTA method and the shortest in the RDO method. EDTA was superior to RDO or HCl in DNA yield and integrity, assessed via DNA extraction, polymerase chain reaction, and silver in situ hybridization using DNA probes. The EDTA method maintained intact nuclear protein staining on immunohistochemistry, while the HCl method produced poor quality images. Staining after the RDO method had equivocal results. RNA in situ hybridization using kappa and lambda RNA probes measured RNA integrity; the EDTA and RDO method had the best quality, followed by HCl. CONCLUSIONS: The EDTA protocol would be the best in preserving genetic material. RDO may be an acceptable alternative when rapid decalcification is necessary.

Preparation of RNA probe for cd99l2 gene of zebrafish labeled with digoxingenin-UTP / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.</p><p><b>METHODS</b>The cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization.</p><p><b>RESULTS</b>The plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development.</p><p><b>CONCLUSION</b>The antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.</p>

Rastreamento universal para cervicite clamidiana: uma revisão sistemática / Universal screening for chlamydial cervicitis: a systematic review

A revisão sistemática teve como objetivo avaliar a efetividade dos testes de ácido nucleico no rastreio da C. trachomatis. A maioria dos estudos foi localizada via internet, entretanto, alguns deles foram encontrados em revistas que abordavam o tema e mediante contato com especialistas. Os artigos foram selecionados após criteriosa avaliação crítica da força de evidência científica, obedecendo às regras da Associação Médica Brasileira e do Conselho Federal de Medicina, além dos critérios de Irwig, para análise qualitativa dos artigos. A revisão incluiu todos os estudos publicados a partir de 1990 que avaliavam testes de ácido nucleico em mulheres sexualmente ativas, assintomáticas e que tivessem sido submetidas à avaliação clinica e a testes moleculares. Os testes de ácido nucleico que utilizavam sondas de RNA e amplificação de DNA (PCR) foram comparados à cultura (padrão-ouro) com o intuito de determinar se seriam método de diagnóstico adequado para o rastreio da infecção. Após análise qualitativa, foram selecionados 12 estudos, mas não foi possível realizar avaliação quantitativa dos mesmos devido à heterogeneidade dos dados. A efetividade e os benefícios dos testes de ácido nucleico justificam estudos de custo-efetividade, com o intuito de avaliar o impacto do rastreio universal na redução das complicações advindas da infecção clamidiana

This systematic review aims at evaluating the effectiveness of the nucleic acid test for detection of C. trachomatis. Most of the studies were searched electronically and key journals were hand-searched. Further studies were identified in the internet and by contacting experts in the field. The articles were selected after careful critical evaluation of the strength of scientific evidence, according to the rules of the Brazilian Medical Association and the Federal Council of Medicine, besides the Irwig's criteria for qualitative analysis of article The review included all studies published from 1990 onward that evaluated nucleic acid tests in asymptomatic, young and sexually active women that have been subjected to clinical evaluation and molecular testing. The nucleic acid tests taken with the use of probes of RNA and amplification (PCR) were compared to culture (gold standard) in order to determine if a method of diagnosis would be appropriate for screening of infection. After the qualitative analysis, we selected 12 studies; it has not been possible to perform their quantitative evaluation due to the heterogeneity of data. The effectiveness and benefits of DNA testing justify the cost-effectiveness studies in order to assess the impact of universal screening in reducing the complications that arise from chlamydial infection

hTR RNA component as a marker of cellular proliferation in oral lichen planus.

Previously, we have shown that the telomerase RNA component hTR is highly expressed in the epithelium of non-dysplastic Oral Lichen Planus (OLP) lesions (11). We concluded that it is possible that this high expression might be related to the increased cellular proliferation seen in OLP rather than being an indicator of potential malignant transformation. In the present study, and in order to confirm our finding in the previous study that hTR might be a marker for cellular proliferation in OLP, we analysed OLP biopsies known to be positive for RNA component of Telomerase (hTR) for the expression of Ki-67 as a marker for cellular proliferation. Fourteen OLP tissue biopsies known to be positive for telomerase RNA component hTR, were investigated using an immunohistochemical approach to determine the rate of cellular proliferation in OLP, looking at the expression of Ki-67 protein as a marker for cellular proliferation. A statistically significant increase was found between Ki-67 expression in OLP in comparison to normal control buccal mucosa samples. The expression of hTR component in OLP might thus be a marker for cellular proliferation.

In vitro Culture of Hepatitis C Virus (HCV) Using Immortalized Hepatocyte / 대한소화기학회지

BACKGROUND/AIMS: It is essential to develop an in vitro culture model of primary hepatocytes for the study of hepatocellular function and the pathogenesis of hepatitis C virus (HCV) infection. In this study, we have established the immortalized primary human hepatocyte (IPHH) and performed in vitro culture of HCV derived from human patient. METHODS: Primary human hepatocytes were isolated from surgically resected liver tissue and then were immortalized by transfection with the SV40 large T antigen. The characterization of the IPHH during culture was analyzed by immunocytochemistry, RT-PCR, Western blot, ELISA, and soft agar assay. Next, sera and/or liver tissue homogenates from surgically resected liver tissues of patients with HCV infection were inoculated for the culture of HCV in IPHH. After HCV RNA extraction from IPHH and culture media, positive or negative stranded HCV RNA was examined by specific nest RT-PCR. RESULTS: IPHH expressed liver-associated proteins but did not express alpha-fetoprotein. Also IPHH showed ammonia removal activity. With regard to its malignant potential, colony formation in soft agar assay was not observed. Next, positive and negative stranded HCV RNAs in IPHH infected with patient's sera plus liver tissue homogenates were clearly detected whereas those in IPHH infected with only patient's sera were not detected. CONCLUSIONS: These results demonstrated the phenotypic characteristics of IPHH and the feasibility in vitro culture system of HCV infected human samples. This system might be useful for study of pathogenesis of HCV infection or hepatocyte-based applications.

Hibridização in situ com sonda não-radioativa para mRNA: princípios e aplicações em patologia / In situ hybridization with non-radioactive riboprobes: principles and applications in pathology

A técnica de hibridização in situ (ISH) tem sido usada para identificar mRNA (ou DNA) em amostras de tecido de material humano e animal. Embora uma série de protocolos para essa técnica seja utilizada, as descrições não são bem detalhadas. O objetivo deste trabalho é descrever a reação de hibridização in situ em tecido fresco e sua aplicação em patologia, tornando mais compreensível essa técnica tão importante, que possibilita observar a localização tecidual e a expressão temporal e espacial dos transcritos de um determinado gene (mRNA). Resultados de reações com as ribossondas PITX1, SHH e WNT-5A, realizadas em amostras de tecido congelado, são apresentados.

In situ hybridization (ISH) has been used to identify mRNA (or DNA) in fresh tissue samples of humans and animals. Several protocols describing this technique are available, although its description is not usually detailed enough. The present work describes in situ hybridization reaction on fresh tissue in a way to make understandable this important technique, which allows verifying the cellular localization, and the spatial and temporal expression, of gene transcripts (mRNA). Results with PITX1, TGIF, SHH and WNT-5A riboprobes, in fresh tissue samples, are presented.

RNA in situ hybridization for the detection of Feline Immunodeficiency Virus in infected cells.

In the present study, we have applied a novel approach to generate specific digoxigenin- and biotin-labeled RNA probes to detect Feline Immunodeficiency Virus (FIV) gag gene in the FIV-infected feline T-lymphoblastoid cell line (MYA-1). This involved cloning of the FIV gag gene into a PCR Script vector containing both T3 and T7 promoters, followed by amplification of the insert and the two promoter sequences, using specific primer sequences. The FIV RNA probes were more sensitive than FIV DNA probes. This approach should make RNA in situ hybridization more accessible for use in routine diagnosis.

Quantification of HIV-1 RNA load by one-tube-one-step RT PCR and real time PCR assay with TaqMan probe.

OBJECTIVES: To develop a less expensive assay to calculate HIV-1 viral load for use in resource-limited countries. MATERIAL AND METHOD: An In-house One-tube-one-step Viral Load Assay (IOVA) was developed by using real-time PCR-based with TaqMan probe. Primers and probe were designed from the conserved region of sequences from all HIV subtypes. A standard curve was generated from reference virus in various dilutions. IOVA was applied on 105 HIV-positive and 25 HIV-negative samples and compared with the results from ROCHE AMPLICLOR. RESULTS: IOVA measured HIV RNA in the samples ranging from 125 to 2 x 10(6) copies/mL. The coefficient of variation of intra- and inter-assay ranged from 0.68% to 7.89%. The sensitivity, specificity, positive and negative predictive values were 92%, 100%, 100% and 79.5% respectively. The parallel quantitative analysis showed high correlation (r=0.95) between IOVA and AMPLICOR. CONCLUSION: A new HIV-1 viral load assay was developed and validated. It was reliable and less expensive.

Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells.</p><p><b>METHODS</b>The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method.</p><p><b>RESULTS</b>The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation.</p><p><b>CONCLUSIONS</b>These new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.</p>

Dynamic expression pattern of kinesin accessory protein in Drosophila.

We have identified the Drosophila homologue of the non-motor accessory subunit of kinesin-II motor complex. It is homologous to the SpKAP115 of the sea urchin, KAP3A and KAP3B of the mouse, and SMAP protein in humans. In situ hybridization using a DmKAP specific cRNA probe has revealed a dynamic pattern of expression in the developing nervous system. The staining first appears in a subset of cells in the embryonic central nervous system at stage 13 and continues till the first instar larva stage. At the third instar larva stage the staining gets restricted to a few cells in the optic lobe and in the ventral ganglion region. It has also stained a subset of sensory neurons from late stage 13 and till the first instar larva stage. The DmKAP expression pattern in the nervous system corresponds well with that of Klp64D and Klp68D as reported earlier. In addition, we have found that the DmKAP gene is constitutively expressed in the germline cells and in follicle cells during oogenesis. These cells are also stained using an antibody to KLP68D protein, but mRNA in situ hybridization using KLP64D specific probe has not stained these cells. Together these results proved a basis for further analysis of tissue specific function of DmKAP in future.

Diagnóstico laboratorial da infecçäo pela Chlamydia trachomatis: vantagens e desvantagens das técnicas / Laboratory diagnosis of Chlamydia trachomatis infection: advantages and disadvantages of the tests

O objetivo deste artigo é revisar e comentar as vantagens e desvantagens dos diferentes tipos de testes de detecçäo de Chlamydia trachomatis na rotina de laboratórios clínicos, com ênfase nas técnicas de amplificaçäo. A Chlamydia trachomatis é considerada a bactéria sexualmente transmissível mais freqüente em países desenvolvidos e de grande impacto no sistema reprodutivo das mulheres. É o agente causador de doenças do trato urogenital, linfogranuloma venéreo (LGV), tracoma, conjutivite de inclusäo e pneumonia no recémðnascido. Um dos fatores de risco para a infecçäo é a prática sexual entre adolescentes. A recorrência das infecções é comum. Episódios sucessivos de infecçäo aumentam o risco de desenvolver seqüelas e a chance de contrair a infecçäo pelo vírus da imunodeficiência humana. O dignóstico da infecçäo pela Chlamydia trachomatis ainda é crítico, devido à freqüência de infecções assintomáticas. As técnicas de amplificaçäo de ácidos nucléicos permitem utilizar urina para a detecçäo da clamídia, simplificando a coleta. Apresentam maior sensibilidade do que a cultura e do que os testes mais utilizados, como a imunofluorescência direta e o enzimaimunoensaio. A cultura celular, utilizada como padräoðouro, tem especificidade de 100 por cento e sensibilidade de 70 por cento a 85 por cento. De acordo com o Centers for Disease Control (CDC), um diagnóstico é considerado definitivo quando positivo em cultura ou em pelo menos dois testes näoðculturais distintos. Os testes de amplificaçäo säo mais dispendiosos do que os demais testes näoðculturais, mas de menor custo que a cultura

Localization of Porcine Reproductive and Respiratory Syndrome Virus Infection in Boars by In Situ Riboprobe Hybridization

The capability of porcine reproductive and respiratory syndrome virus (PRRSV) to be shed in semen for extended periods of time has been suggested to be a principal factor for viral transmission via insemination. In attempts to gain insights into the mechanism of PRRSV persistence in boars, tissue distribution and sites of viral infection were investigated by in situ hybridization (ISH) using digoxigenin-labeled RNA probe and the ISH results were compared with those of reverse transcription-nested polymerase chain reaction (RT-nested PCR). Animals were intranasally inoculated with 104 median tissue culture infectious dose of PRRSV VR-2332 and tissues collected at different times were examined. At day 7 postinfection, limited number of hybridization positive signals was observed in cells within or between seminiferous tubules in the testis sections while relatively abundant hybridization positive signals were observed in the brain stem and tracheobronchial lymph node. At later days of infection, hybridization positive signals were observed in cells within seminiferous tubules with much reduced frequency. Lack of agreement with the RT-nested PCR assay results in testis tissues obtained at days 14, 28, and 59 postinfection suggested that PRRSV infection in the testis may be extremely restricted, and may not necessarily constitute a major viral source in semen during extended periods of seminal shedding.

The in situ hybridization effort estimation between antisense RNA probe and random primed DNA probe / 法医学杂志

OBJECTIVE@#To estimate the effort between antisense RNA probe and random primed DNA probe.@*METHODS@#The in situ hybridization was conducted on parafin section from wounding model of rat skin.@*RESULTS@#Although both probes appeared positive staining, RNA probes was superior to DNA probes in terms of depth of staining and background.@*CONCLUSION@#RNA probe showed more satisfactorily on ISH.

El colágeno de la reestenosis post angioplastia con stent: ¿se origina en la íntima o en la adventicia? / Origin of collagen in restenosis post stenting: intima or adventitia?

Background: Restenosis post stenting is due to the deposit of extracellular matrix, mainly collagen in the neointima. Controversy exists regarding if collagen is generated locally or by immigration from the adventitia. Aim: To study the fibrocellular response after stent implantation in rabbit iliac arteries. To observe, by immunohistochemistry and in situ hybridization, if collagen type I mRNA is expressed in the neointima, in the media or in the adventitia. Material and methods: Thirty eight white rabbits (New Zealand) of 4 kg received an hypercholesterolemic diet during 1 month. After this period, in all but 6 of them, an angioplasty with stent implantation was performed via right carotid artery in both iliac arteries, using a 1:1.3 relationship regarding the reference vessel. Angiograms were performed at day 0, 4, 21, and 40, followed by paraffin fixation of the injured segments, immunohistochemistry for a-actin and in situ hybridization to detect procollagen type I (a1R1) mRNA. Results: No hybridization was observed in non injured arteries or at day 0 (n= 6). Expression of a1R1 mRNA was observed in the neointima starting at day 4 after stenting (n= 8). At day 21 (n= 8) hybridization of procollagen type I was not only observed in the neointima, but also in the media, which became equally intense in both areas. At day 40 (n= 6) hybridization was observed similarly in the media and adventitia. Conclusions: In this model, hybridization of procollagen type I started in the neointima, then involved the media and finally the adventitia. This finding might be useful for designing therapies to be delivered locally at the end of an angioplasty to prevent collagen deposition in the neointima

Improved Technique of Digoxigenin Labeled RNA in situ Hybridization

BACKGROUND: A practical RNA in situ hybridization method using digoxigenin labeled RNA probes is described in order to evaluate the technical difficulties and problems in RNA in situ hybridization. METHODS: The paraffin sections, routinely processed in the Pathology Laboratory, were tested for the possibility of RNA in situ hybridization instead of the RNase free paraffin sections, fixed in 4% paraformaldehyde and prepared using RNase protection procedures. RESULTS: Most of the paraffin sections, fixed in 10% neutral formalin solution in fresh condition, showed relatively good reaction of RNA in situ hybridization, although the necrotic tissue and autopsy specimens showed poor reaction of RNA in situ hybridization. A refixation procedure using a 4% paraformaldehyde solution was evaluated for optimal expression of mRNA in the paraffin sections. CONCLUSION: The treatment of 4% paraformaldehyde before the treatment of proteinase K showed better in situ hybridization than did the treatment of 4% paraformaldehyde after the treatment of proteinase K. Also a new Polymerase Chain Reaction (PCR)-based method of RNA probe production showed consistently good results.

In Situ Detection of mRNA and RNA Component of Human Telomerase in Proliferative Lesions of the Stomach

BACKGROUND: Proliferative lesions of the stomach were investigated by in situ hybridization using RNA probes for telomerase components and compared with the results by TRAP (telomeric repeat amplification protocol) assay. METHODS: RNA probes for hTR (human telomerase RNA component) and hTERT (mRNA coding for a catalytic subunit of human telomerase) were made by cloning and in vitro transcription. The probes were applied for in situ hybridization in 23 cases of adenocarcinoma of the intestinal type and adjacent dysplasia, and in the normal and metaplastic mucosa of the stomach. RESULTS: Telomerase activity by TRAP was positive in all cases of adenocarcinoma, most cases of dysplasia, and many cases of normal mucosa. hTR in situ hybridization showed positive staining in the adenocarcinoma cells, dysplastic cells, a few cells in the proliferation zone of the normal mucosa, and a few infiltrated lymphocytes. hTERT showed positive staining in the same cells. CONCLUSIONS: Telomerase is expressed in most cases of dysplastic lesions and is thought to be acquired in the early steps of carcinogenesis. The expression is noted in a few cells of the normal proliferative zones and the infiltrated lymphocytes, emphasizing the importance of in situ detection of telomerase at the cell level.

Study of gene probes in childhood leprosy.

The objective was to detect nucleic acids of M. leprae in skin lesions of leprosy patients and study the effect of treatment on these nucleic acids, using r-RNA gene probes, using a cross sectional study. The study was carried out at Department of Paediatrics, S.N. Medical College, Agra and Department of Microbiology, Central JALMA Institute for leprosy, Agra. The study included 32 cases of leprosy less than 16 years of age, divided into 3 groups viz. without treatment (12 cases), in middle of treatment (11 cases) and at the end of treatment (9 cases). All cases were subjected to a detailed history and thorough clinical examination. All of them had smear examination and lepromin test done and their skin biopsies were subjected to gene detection. Nucleic acids were isolated from skin biopsies of all cases by standard procedure. After dot blotting of these nucleic acids, they were hybridised with radioactive (p32) r-RNA probes. The results were interpreted after getting the X-ray films processed with background signals from controls. Majority of cases were between 13-16 years of age. As age advanced, the disease moved from tuberculoid end of spectrum towards lepromatous end (p < 0.05). Majority of paucibacillary (PB) cases were lepromin positive while majority of multibacillary (MB) cases were lepromin negative (p < 0.05). In specimens of untreated cases, 50% of PB specimens and 87.5% of MB specimens were positive for r-RNA probes. In multibacillary type 100% smear positive specimens and 67% smear negative specimens were positive for r-RNA probes. In patients during the middle of treatment positivity for r-RNA decreased and 20% of PB specimens and 16.6% MB specimens were positive. At the end of treatment (1 year for PB cases and 2 years for MB cases), the results of r-RNA were negative, which indicated that the treatment regimens used in the study were effective. This study supports the usefulness of r-RNA probes as a diagnostic and therapeutic tool in childhood leprosy.

Expression of GnRH-receptor mRNA in the Placenta during Pregnancy / 대한산부인과학회잡지

OBJECTIVE: Our previous study demonstrated that the placental GnRH and GnRH mRNA did not parallel the time course of hCG secretion, though it is thought to be one of the potential paracrine regulators of hCG secretion from the trophoblasts. The present study was designed to examine the potential variation in GnRH-receptor mRNA expression in the placenta, which may account for the GnRH-mediated action of hCG secretion during pregnancy. METHODS: Human placentas in firt, second, and third trimester of normal pregnancy were obtained. These placentas were fixed with 4% paraformaldehyde and embedded in OCT compound, and sectioned by cryostat. For in situ hybridization, S labeled RNA probes were used and followed by autoradiography. RESULTS: The GnRH-receptor mRNA signals were present in both cytotrophoblast and syncytiotrophoblast cell layers. Signal intensities varied with gestational ages and were abundant at 6-7 weeks, peaked at 9-12weeks, declined at 14 and 24 weeks, and were barely detectable at term. The present study demonstrates that GnRH-receptor mRNA exhibits changes paralleling the time course of hCG secretion during pregnancy CONCLUSION: These data provide mechanistic understanding that the paracrine/autocrine regulation of hCG secretion by placental GnRH is mediated through an increase followed by a decline in GnRH-receptor mRNA expression from the first trimester to term placenta.

Developmental mRNA Expression of Cellular Retinoic Acid Binding Protein l and l in Rat

PURPOSE: Retinoic acid (RA) is well known as a potent teratogenic agent in both deficiency and excess. Cellular retinoic acid binding proteins (CRABPs) are involved in RA. We carried this study in order to determine the possible relations of CRABPs with RA-induced teratogenesis through observation of the expression patterns of CRABP l and ll in developing rats. METHODS: 35S-labeled RNA probes were synthesized using SP6-RNA polymerase in CRABP l and T7-polymerase in CRABP ll. The distribution of CRABP l and ll transcripts analyzed by in situ hybridization of rat embryosections from day 12 to 19, and postnatal brains from day 1 to 14. RESULTS: The CRABP ll transcripts were more widely distributed than CRABPl distribution, however, the relative level of CRABPl transcripts were higher than CRABP ll. The CRABP l mRNA transcripts showed its highest expression on the 16th day of gestation and these distribution correlated well with structures known to be targets of RA-induced teratogenesis. CRABP ll transcripts were expressed in brain vesicle, spinal cord, head, face, tongue and genital tubercle and also found in the structures which are not involved in RA-induced teratogenesis. CONCLUSION: These results suggest the possible involvement of both CRABPs in the RA-induced teratogenesis. However, CRABP l may have more specific roles than CRABP ll which may play a role through a different mechanism.

Occurence of citrus viroids in Costa Rica

A survey for citrus viroids was conducted in the major citrus commercial growing areas in Costa Rica. Screening of 36 sweet orange and 12 lemon trees resulted in the detection of members of four of the five citrus viroid groups as determined by nucleic acid hybridization using specific RNA probes and polymerase chain reaction (PCR) using specific oligonucleotide primers. CEVd, CVd-IIa, CVD-IIb and CVd-III viroids were found to be widespread in the three main regions of commercial citrus production. CVd-Ib was only found in lemon in Nicoya