Nested Primer Polymerase Chain Reaction Research Articles

Detection of "Candidatus Liberibacter asiaticus" Using Conventional PCR and Nested-PCR and Identification of Primer Sensitivity through Q-PCR in HLB Infected Mandarin Orange and Acid Lime Plants in Tamil Nadu

One of the most destructive citrus diseases in the world is called citrus Huanglongbing (HLB). For effective HLB associated ‘Candidatus liberibacter spp.’ prevention, sensitive and precise diagnostics are essential. Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (CLam) are among the ‘Candidatus liberibacter spp.’ that infect citrus. The most common of these is the species known as Las. To assess the impact of citrus greening disease in the citrus orchard, leaf samples exhibiting greening symptoms were picked up from 210 mandarin orange trees from nine localities in four districts and among them 189 plants showed HLB positive and leaf samples are picked up from 216 acid lime trees from nine localities in six districts of Tamil Nadu where citrus is cultivated in a larger area and among them 202 plants showed HLB positive. A “conventional singleplex polymerase chain reaction (PCR) Nested PCR and Q-PCR (Quantitative PCR)” was performed on the extracted DNA samples to detect Ca. Liberibacter asiaticus. Among all the mandarin orange growing localities from “Kanalkadu and Thandikudi in Dindigul district” and among the acid lime fields, the samples from “Sankarankoil from Tirunelveli district and Kallar from Nilgiris district” showed consistent amplification of 1160 base pairs fragment by using specific primers OI1 and OI2C and also 703 base pairs fragment with gene-specific primers A2 and J5 targeting beta-operon (rpiKAJL-rpoBC) gene which is a ribosomal protein gene of Ca. Liberibacter asiaticus, these results showed that these districts are exclusively positive to Clas . The nested PCR method significantly increased the sensitivity to identify Las up to 10 times and 100 times, respectively, compared to qPCR and conventional PCR in this study. A set of nested PCR primer pairs were examined to diagnose Las. Nested PCR was used to analyse 14 samples from 2 different citrus cultivars in 2 different districts. Among those again kanalkadu orchard for mandarin orange and Sankankoil orchard for acid lime showed more disease incidence and consistent amplification. The findings indicate that all the samples tested positive for HLB; the samples of blotched and chlorotic leaves from the acid lime field at Sankarankoil (thirunelveli district) and mandarin orange field at kanlakaadu (Dindigul district) had the highest positive detection rate (100%). The outcomes show that the nested PCR primer pairs are capable of detecting Las in a variety of symptomatic tissues, citrus cultivars, and geographical locations. The set of nested PCR primers used in the current work will be a very helpful addition to the existing methods for Las detection.

Development of an ultra-sensitive single-tube nested PCR assay for rapid detection of Campylobacter jejuni in ground chicken

Traditional culture-based detection methods for Campylobacteri jejuni, a leading cause of human bacterial gastroenteritis worldwide, are time-consuming, cumbersome, and lacking in reliability. While polymerase chain reaction (PCR) has been frequently used for pathogen testing, it might generate false-negative results due to inadequate sensitivity. This study was the first to explore novel single-tube nested PCR (STN-PCR) to detect pathogens in food. Two pairs of nested PCR primers were designed based on the hippuricase gene of C. jejuni. The annealing temperatures and concentrations of nested primers were optimized to ensure the sequential use of outer and inner pairs of primers during amplification. Efficacy of the developed STN-PCR assay was compared with standard culture-based methods and conventional PCR in artificially contaminated ground chicken homogenate. Limit of detection of the STN-PCR assay was determined to be 3.6 × 101 CFU/ml of C. jejuni in the homogenate without enrichment, which was 100 times lower than conventional PCR. Moreover, 0.1 CFU/g of C. jejuni in ground chicken homogenate was identified by STN-real time PCR (rtPCR) after 24 h of enrichment, while a 48-h enrichment was required for culture-based methods and conventional rtPCR. This developed assay provides a powerful tool for rapid, highly specific, and ultra-sensitive detection of C. jejuni and may potentially be used to identify contaminated chicken and other foods.

PCR Markers Derived from Comparative Genomics for Detection and Identification of the Rice Pathogen Ustilaginoidea virens in Plant Tissues.

Ustilaginoidea virens is a fungal pathogen that causes the false smut disease in rice and reduces the yield and quantity of the grains. A nested polymerase chain reaction (PCR)-based assay was developed to detect U. virens using genes of U. virens as specific targets. Ninety-six candidate genes of U. virens were found through first-round homology screening against a local database comprising 46 genomes of fungi, bacteria, and plants, with a second-round comparison with the GenBank NR database to further identify genes unique to U. virens. Among 96 remaining candidate genes, 20 of them (GenBank accessions KY617806 to KY617825) were randomly selected for further testing and, eventually, six sets of nested PCR primers were developed after further sensitivity, specificity, and detection tests. All six sets could detect DNA of U. virens at as little as 1 to 10 fg/μl from field or lab samples. These primers may be used to detect infection by U. virens at early stages, for use in research toward mitigating disease spread, as well as for studying the ecology of U. virens. This study also serves to illustrate that a comparative genomics method may allow for selection and development of highly specific primers once draft or complete genomes are available.

Evaluation of highly conserved hsp65-specific nested PCR primers for diagnosing Mycobacterium tuberculosis.

To evaluate the sensitivity and specificity of a new nested set of primers designed for the detection of Mycobacterium tuberculosis complex targeting a highly conserved heat shock protein gene (hsp65). The nested primers were designed using multiple sequence alignment assuming the nucleotide sequence of the M. tuberculosis H37Rv hsp65 genome as base. Multidrug-resistant Mycobacterium species along with other non-mycobacterial and fungal species were included to evaluate the specificity of M. tuberculosis hsp65 gene-specific primers. The sensitivity of the primers was determined using serial 10-fold dilutions, and was 100% as shown by the bands in the case of M. tuberculosis complex. None of the other non M. tuberculosis complex bacterial and fungal species yielded any band on nested polymerase chain reaction (PCR). The first round of amplification could amplify 0.3 ng of the template DNA, while nested PCR could detect 0.3 pg. The present hsp65-specific primers have been observed to be sensitive, specific and cost-effective, without requiring interpretation of biochemical tests, real-time PCR, sequencing or high-performance liquid chromatography. These primer sets do not have the drawbacks associated with those protocols that target insertion sequence 6110, 16S rDNA, rpoB, recA and MPT 64.

Abstract 3186: Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR

Abstract Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. To increase detection sensitivity of FLT3-ITD, we developed new detection algorithm using next generation sequencing (NGS) data. We validated results using nested polymerase chain reaction (PCR) methods. We compared results of NGS data, nested PCR and conventional PCR methods. First, using whole exome sequencing data of 83 AML patients, we applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped. Second, we deigned two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was deigned to target previously reported regions which are frequently occurred ITD mutation. PCR reactions of two steps were performed using the PCR primers sequentially. In these 83 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, this resulted in positive FLT3-ITD in 11 patients (11/83, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all of 11 patients. Nested PCR detected additional 4 patient positive for FLT3-ITD in this population. For 68 patients, FLT3-ITD was negative by both NGS and nested PCR method. Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population. And the concordance rate of NGS method and nested PCR was 95.2% (79/83). Then we investigated clinical significance of sensitive FLT3-ITD detection. For this, we performed nested PCR and conventional PCR at the same time in 238 AML patients to detect FLT3-ITD. Positive rate for FLT3-ITD was 20% (48/238) and 10% (24/238) by nested PCR and conventional PCR respectively. When survival analysis was performed, among patients with negative FLT3-ITD result by conventional PCR, patients who showed positive for FLT3-ITD by nested PCR had shorter overall survival compared to those who showed negative for FLT3-ITD by nested PCR. (p = 0.03). This implies that sensitive FLT3-ITD detection using nested PCR is clinically meaningful. Diagnosis of FLT3-ITD is very important genetic factor, leading a therapeutic direction for AML patient. Here we report that we have developed alternative more sensitive detection methods for FLT3-ITD based on nested PCR and NGS. Sensitive detection of FLT3-ITD was clinically meaningful, suggesting that these methods should be incorporated in a future clinical practice. Also, we want to note that, NGS method is capable of quantifying FLT3-ITD size and amount in AML patients. Citation Format: Daeyoon Kim, Yoojin Hong, Youngil Koh, Sung-Soo Yoon, Choong-Hyun Sun, Kwang-Sung Ahn, Seungmook Lee, Hongseok Yun, Suyeon Lee. Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3186.

HBV Genotyping and Phylogenetic Analysis through Multiplex PCR Using Type Specific Primer Pairs in Pakistani Population

Background: Hepatitis B virus causing acute and chronic hepatocellular carcinoma relics a serious health issue affecting more than 350 million people worldwide. Geographically, nine genotypes of HBV have been identified so far. Four open reading frames named (ORF P, S, F and X) renders it to specifically targets hepatocyte cells. Additionally, reverse transcription process present in the life cycle of HBV caused four order higher mutation rates as compared to adenovirus. The structural and functional differences between HBV genotypes are the mainstay to severity, complications, treatment and possibly vaccination against the virus. Hepatitis B genotyping is important for assessing its clinical implications and geographical distribution, but the sub-genotypes have been found useful for determination of specific genomic markers related to hepatocarcinogenesis and medical therapies. In Pakistan, there is no reported data available on molecular evolutionary analysis of HBV. A study was, therefore, much needed to evaluate the spectra of dominant strains prevalent here. Objectives: This study was aimed to figure out the prevalence of HBV genotypes among population in Pakistan. In this research, we focused to find out the new strains and sub genotypes of HBV. Pakistan is one of the HBV endemic countries but there is no data on subgenotypes and their recombination or the phylogenetic relatedness of the virus endemic in the country. The present study was carried out in this perspective. This study will help future researcher to study some other aspects of HBV related strains in Pakistan and also about its medical therapies. Place and duration of study: The present study was carried out at Polymerase Chain Reaction (PCR) lab, Nuclear Medicine, Oncology and Radiotherapy Institute [NORI], Islamabad, and the samples were collected between September 2012 to February 2014. Study design: A total of 450 Hepatitis B surface antigen HBsAg and HBV DNA positive samples were collected in the first step during the period of September 2012 to February 2014 and sent to Nuclear Medicine Oncology and Radiotherapy Institute [NORI], for further analysis. All the samples were genotyped by type specific nested PCR primer pair method for 8 HBV genotypes from A through H. The patients were randomly selected irrespective of their age and gender and a written consent (parental consent in case of less than 18 years of age) was obtained. The study was approved by ethical review committee. In our research, we have used the type specific primer technique to find out the dominant strain in Pakistani populations and furthermore its phylogenetic analysis with reference sequences retrieved from NCBI databases. Results: Our results showed a direct relationship between viral load and identified genotypes and pinpointed that genotype D is the most pre-dominant genotype and prevailing in high proportions among all genotypes in Pakistani population. Mix HBV genotype infections of genotype A with B & C, D with B and C constitute about 17 percent of all the samples. Subgenotypes of different HBV strains have been identified on the basis of differences in the complete nucleotide sequences. Positive PCR results were repeated twice for confirmation. Furthermore, phylogenetic analysis revealed a homology between our reported sequences and reference sequences. The phylogenetic method showed that genotypes/sub-genotypes varies in the geographical areas and correlate strongly with ethnicity. Conclusion: Genotype D was identified and come out to be the most dominant genotype in Pakistani community showing sub-genotypes of D1 & D3 in our study. Genotype A and D are present as co-infection with each other and contributed as the second prevailing genotypic group. Additionally, phylogenetic analysis revealed that genotypes varies in the geographical areas and correlate strongly with reference sequences from NCBI.

감자T바이러스 검정을 위한 RT-PCR 및 Nested PCR 진단시스템 개발

Potato virus T (PVT) is a plant pathogen in the family Betaflexiviridae, group IV single-stranded positive sense RNA viruses. The major host of PVT is potato, and it has been reported in Ullucus tuberosus, Oxalis tuberosa and Tropaeolum tuberosum. This study aimed at developing reverse transcription (RT)-polymerase chain reaction (PCR) and nested PCR techniques for specific detection of PVT. Finally, Two RT-PCR primer sets were developed and verified. The RT-PCR products were amplified to 734 (PVT RT-PCR primer set 6) and 828 bp (PVT RT-PCR primer set 29) long to detect PVT. The nested PCR primer sets [PVT-N70/C20 (734→315 bp) and PVT-N75/C30 (828→529 bp)] were developed which are high sensitivity and verification for detection of PVT. Furthermore, a modified-positive control plasmid is use to verify contamination of laboratory in PVT detection. This study supported the diagnose PVT in potato or PVT related hosts.

Detection and phylogenetic characterization of Columbid circoviruses in Chaharmahal va Bakhtiari province, Iran

The virus genus Circovirus belongs to the family Circoviridae and contains virus species with circular single-stranded DNA genomes. The viruses are known to infect vertebrate species, including pigs, dogs, pigeons and ducks. In this study a nested polymerase chain reaction (PCR) was applied to investigate prevalence of circoviruses in pigeons in the Chaharmahal va Bakhtiari province, Iran. A total of 50 faecal samples were subjected to nucleic acid extraction, PCR amplification and DNA sequencing. Nested PCR primers were designed to amplify a 508 base pair segment of the pigeon circovirus (PiCV) capsid gene. Of the 50 faecal samples examined, 12 (24%) produced the expected DNA amplicons with identical DNA sequences. Phylogenetic analysis has grouped the viruses with those classified as group A circoviruses. The viruses were closely related to PiCVs found in Poland, Northern Ireland, the USA, Nigeria and Hungary. To the authors' knowledge, this is the first report of molecular detection and genomic characterization of PiCV from Iran.

First Report of Phytoplasma (16SrI) Associated with Yellow Decline Disease of Royal Palms [Roystonea regia (Kunth) O. F. Cook] in Malaysia

AbstractRoyal Palms (Roystonea regia) with symptoms such as severe chlorosis, stunting, collapse of older fronds and general decline were observed in the state of Selangor, Malaysia. Using polymerase chain reaction (PCR) amplification with phytoplasma universal primer pair P1/P7 followed by R16F2N/R16R2 and fU5/rU3 as nested PCR primer pairs, all symptomatic plants tested positively for phytoplasma. Results of phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences revealed that the phytoplasma associated with Royal Palm yellow decline (RYD) was an isolate of ‘Candidatus Phytoplasma asteris’ belonging to a new 16SrI‐subgroup. These results show that Roystonea regia is a new host for the aster yellows phytoplasma (16SrI). This is the first report on the presence of 16SrI phytoplasma on Royal Palm trees in Malaysia.

Short Communication: HIV Type 1 Tropism Determination in a Novel Dried Blood Spot Membrane and the Use of a Mixture of Outer Nested Polymerase Chain Reaction Primers

Genotropism was determined in 608 Brazilian samples collected in dried blood spots using Polyethersulfone collection cards. Patients were infected by subtype B (88.8%), F (5.6%), C (3.3%), A (1.8%), and G (0.5%). All patients were exposed to three classes of antiretrovirals, and 59.8% of the samples harbored R5 viruses, 35% non-R5-tropic viruses, and 5.1% harbored mixtures of R5 and non-R5-tropic viruses, with non-R5 more prevalent among clade B-infected patients as compared to non-B (42.8% versus 19.1%; p<0.0003). A strategy using a mixture of outer nested polymerase chain reaction (PCR) primers reduced the number of negative PCR results from 39% to 19%.

Rapid Detection of Red Turpentine Beetle (Dendroctonus valensLeconte) Using Nested PCR

Abstract The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Curculionidae), which is native to North America, is an invasive wood-boring insect species in China with disastrous consequences. Plant quarantine is an important method to hinder the invasion of this species. In order to distinguish RTB from the other four closely related bark beetles, Scolytus schevyrewi Semenov, Xyleborus artecomans Schedl, Xyleborus interjectus Blandford and Blastophagus minor Hartig, which are frequently intercepted at Chinese ports, we developed a nested polymerase chain reaction (PCR) assay. The specificity of the nested PCR primers to recognize RTB was confirmed by the failure to amplify genomic DNA from the other four Scolytinae species. The sensitivity was approximately 40 pg of a recombinant RTB plasmid. We evaluated the validity of the assay by analyzing bark beetles intercepted at quarantine stations. Our findings demonstrate that this specific molecular method allows for the rapid identificat...

Comparative Analysis of Polymerase Chain Reaction Assay for Herpes Simplex Virus 1 Detection in Tear

PurposeTo comparatively analyze the methodological efficacy of the polymerase chain reaction (PCR) assay for herpes simplex virus 1 (HSV) detection in tears.MethodsThis retrospective study reviewed the medical records of 115 patients who were clinically diagnosed with herpes keratitis, and their tear samples were collected for HSV detection. PCR positive rates were analyzed for their dependence on the PCR primers used (conventional PCR primer vs. nested PCR primer), the tear collecting method used (micropipetting vs. collection with schirmer strip), the disease manifestation and the patient's previous medication history.ResultsHSV DNA was detected in 23 out of 115 (20%) tear samples. The PCR positive rate in tear samples did not differ depending on the PCR primer or tear collection method used. Typical epithelial lesions showed a higher positive rate (31.4%) than atypical epithelial lesions (10.9%). The previous history of the antiviral agent seemed to affect the PCR positive rate.ConclusionsAlthough the PCR positive rate was not dependent on the tear collection method or primers, HSV detection in tears using PCR was shown to be a supplementary diagnostic test in typical and atypical herpes epithelitis.

Identification of ‘Candidatus Phytoplasma solani’ Associated with Tree Peony Yellows Disease in China

AbstractTree peony (Paeonia suffruticosais) plants with yellowing symptoms suggestive of a phytoplasma disease were observed in Shandong Peninsula, China. Typical phytoplasma bodies were detected in the phloem tissue using transmission electron microscopy. The association of a phytoplasma with the disease was confirmed by polymerase chain reaction (PCR) using phytoplasma universal primer pair R16mF2/R16mR1 followed by R16F2n/R16R2 as nested PCR primer pair. The sequence analysis indicated that the phytoplasma associated with tree peony yellows (TPY) was an isolate of ‘Ca. Phytoplasma solani’ belonging to the stolbur (16SrXII) group. This is the first report of a phytoplasma associated with tree peony.

Detection of human papillomavirus (HPV) L1 gene DNA possibly bound to particulate aluminum adjuvant in the HPV vaccine Gardasil®

Medical practitioners in nine countries submitted samples of Gardasil® (Merck & Co.) to be tested for the presence of human papillomavirus (HPV) DNA because they suspected that residual recombinant HPV DNA left in the vaccine might have been a contributing factor leading to some of the unexplained post-vaccination side effects. A total of 16 packages of Gardasil® were received from Australia, Bulgaria, France, India, New Zealand, Poland, Russia, Spain and the United States. A nested polymerase chain reaction (PCR) method using the MY09/MY11 degenerate primers for initial amplification and the GP5/GP6-based nested PCR primers for the second amplification were used to prepare the template for direct automated cycle DNA sequencing of a hypervariable segment of the HPV L1 gene which is used for manufacturing of the HPV L1 capsid protein by a DNA recombinant technology in vaccine production. Detection of HPV DNA and HPV genotyping of all positive samples were finally validated by BLAST (Basic Local Alignment Search Tool) analysis of a 45–60 bases sequence of the computer-generated electropherogram. The results showed that all 16 Gardasil® samples, each with a different lot number, contained fragments of HPV-11 DNA, or HPV-18 DNA, or a DNA fragment mixture from both genotypes. The detected HPV DNA was found to be firmly bound to the insoluble, proteinase-resistant fraction, presumably of amorphous aluminum hydroxyphosphate sulfate (AAHS) nanoparticles used as adjuvant. The clinical significance of these residual HPV DNA fragments bound to a particulate mineral-based adjuvant is uncertain after intramuscular injection, and requires further investigation for vaccination safety.

Probable Blastomyces dermatitidis Infection in a Young Rat

A 21-week-old male untreated control SHR/NCrlNarl rat was found dead during an experiment. Grossly, pulmonary lesions were characterized by multifocal to coalescing firm gray-white nodules randomly scattered on the surface. Microscopically, bronchopneumonia was found with pyogranulomas containing neutrophils, macrophages, and numerous thick-walled yeast cells. Yeast cells, 5 to 25 μm in diameter, with no branching of hyphae were observed by staining with hematoxylin and eosin, Diff-Quik, and periodic acid-Schiff. Furthermore, polymerase chain reaction (PCR) using panfungal and nested PCR primers were used for detection of Blastomyces dermatitidis DNA in the lung tissue. After sequencing and matching with DNA sequences in the GenBank, the sample showed a similarity of 94.6% and 97% to Ajellomyces dermatitidis (B. dermatitidis), respectively. On the basis of these results, probable pulmonary blastomycosis was diagnosed. The origin of the infection in the colony rat is undetermined.

Increased Sensitivity and Specificity ofBorrelia burgdorferi16S Ribosomal DNA Detection

The DNA of Borrelia burgdorferi spirochetes extracted by ammonium hydroxide was used as the template for nested polymerase chain reaction (PCR) amplification of the species-specific 16S ribosomal DNA (rDNA). The primers were those well known to be specific for signature sequence amplification of the B burgdorferi sensu lato 16S ribosomal RNA gene. The positive 293-base-pair nested PCR amplicon was subjected to routine direct automated Sanger sequencing. A 50-base sequence excised randomly from the sequencing electrophoretogram between the 2 nested PCR primer binding sites was sufficient for the Basic Local Alignment Search Tool (BLAST) analysis to validate the B burgdorferi sensu lato 16S rDNA without a reasonable doubt. Nested PCR increased the sensitivity of DNA detection by 100- to 1,000-fold. DNA sequence validation based on BLAST algorithms using the GenBank database practically eliminates any possibility of false-positive results due to molecular misidentification. This technology may be a valuable supplement to the current serologic tests for Lyme disease.

Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing for detection of avian reovirus

Avian reovirus (ARV) causes several disease syndromes in poultry including arthritis, malabsorption syndrome and chronic respiratory disease that result in major economic losses. Early detection is very important for the control of the ARV-induced infections. This study was therefore aimed at developing a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) assay for detection of ARV. This assay combines nested polymerase chain reaction (PCR) and magnetic bead-based DNA probing systems greatly increasing its sensitivity and specificity. Alignment of ARV S2 gene from different ARV genotypes and serotypes was done to find the highly conserved regions for primer and probe design. Two reverse transcription (RT)-PCR primer pairs, six nested PCR primer pairs, and one magnetic probe were tested to find the most specific ones for ARV detection. The optimal conditions for RT-PCR, nested PCR, and hybridization of magnetic probe were established. The optimal annealing temperatures for RT-PCR and nested PCR were 62.1 and 54.8 °C, respectively. The optimal hybridization temperature was 51.2 °C using hybridization buffer (5× SSC and 0.5% SDS). The sensitivity of the kit was 5 copies/μl of ARV genomic RNA. The kit was very specific as all negative controls failed to show any positive reactions. The kit shows good reproducibility with intra- and inter-assay coefficient of variation (CV) of 1.3 and 1.7%, respectively. In addition, diferent serotypes and genotypes of ARV were tested by RAPID-BAP assay to estimate the practicability of the kit in clinical samples. All of ARV serotypes and genotypes tested could be detected by this kit proving that the kit is suitable for clinical application.

128 MITOCHONDRIAL DNA DELETIONS IN RHESUS MACAQUE OOCYTES, EMBRYOS, AND ADULT AND EMBRYONIC STEM CELLS

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Previous data have shown that mitochondrial DNA (mtDNA) deletions are present both in human oocytes and in embryos from in vitro fertilization (IVF) patients and suggest that accumulation of these deletions may contribute to mitochondrial dysfunction and impaired ATP production. In addition, high levels of mitochondrial mutations are present in skeletal muscle fibers from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in non-human primate oocytes and embryos generated by IVF and to determine whether mtDNA mutations are already present in immature oocytes from rhesus ovaries. Using a nested primer polymerase chain reaction (PCR) strategy, we determined the frequency of the rhesus common deletion in immature oocytes compared with stimulated oocytes and embryos. There was a low incidence (21%) of the rhesus common deletion present in immature, unstimulated oocytes derived from necropsied ovaries of 2 to 10-yr-old rhesus macaques. However, there was &gt;3-fold increase (71.4%) in the frequency of deleted mtDNA in stimulated oocytes and IVF embryos from age-matched fertile monkeys. We postulated that, in addition to skeletal muscle, a similar time-dependent accumulation of mtDNA deletions occurs in fertile rhesus macaque oocytes and embryos. We are now investigating the effects of culture and passage number on mtDNA deletions in primate adult and embryonic stem cells. We propose the rhesus monkey to be an excellent model to assess the quality of gametes and embryos, as well as stem cells, and their developmental competence in human and non-human primates. This study was supported by National Institutes of Health grants RR15395 and HD045966.

A New Y-Chromosome-Specific Marker for Pacific Salmon

A genetic marker showing Y-chromosome-specific linkage in Chinook salmon Oncorhynchus tshawytscha, chum salmon O. keta, and coho salmon O. kisutch has been recovered by amplified fragment length polymorphism screening of haploid Chinook salmon embryos produced by androgenesis. Nested polymerase chain reaction primers designed from this sequence show sex-specific amplification. This region includes an apparent pseudogene of an open reading frame found autosomally in all Pacific salmonids tested. The test is not successful for rainbow trout O. mykiss or pink salmon O. gorbuscha but shows promise for use with sockeye salmon. Our approach should be applicable to isolating sex-specific sequences in other fish species. This marker (OtY2-WSU) should have applications in salmonid aquaculture, in studies of Y chromosome evolution, and in investigating possible sex reversal events in Pacific salmon.

Studies of the molecular basis of IgA production, subclass regulation and class-switch recombination in IgA nephropathy patients.

IgA nephropathy (IgAN), the most common form of glomerulonephritis, is characterized by normal to elevated levels of serum IgA. In order to understand the molecular mechanism(s) involved in the production of IgA in IgAN, peripheral blood mononuclear cells (PBMC) from these patients were analysed in this study. IL-10, transforming growth factor-beta 1 (TGF-beta 1) and CD40 have previously been shown to be involved in IgA production. We show here that CD40L expression was increased three-fold in these patients. However, expression of TGF-beta 1 in serum levels was comparable to controls. In vitro stimulation of PBMC with a polyclonal activator resulted in a three-fold increase in synthesis of both IgA subclasses, with a preference for IgA1 RNA. In situ hybridization studies also showed a three-fold increase in the numbers of IgA1- and IgA2-producing cells, but the subclass distribution was similar to the controls. Furthermore, using the nested primer polymerase chain reaction (PCR) for amplifying switch (S mu/S alpha) breakpoints we could demonstrate that in unstimulated PBMC the switch frequency did not differ from that of control donors. Sequence analysis of the amplified switch breakpoints and the I alpha regulatory region from patients showed no structural abnormality. Although we have previously demonstrated a correlation to in vivo germ-line RNA expression and class switching, no I alpha transcripts were detected in unstimulated PBMC from these patients. However, stimulation of PBMC with TGF-beta 1 resulted in I alpha production. Taken together, results from in vivo and in vitro studies suggest that increased cytokine production and hyperresponsiveness to polyclonal stimulation may play an important role in the increased synthesis of IgA. The preference for IgA1 is due to increased production of IgA1 per cell, and the absence of I alpha RNA indicates that additional defect(s) in immune regulation may play an important role in the pathogenesis of IgAN.