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PubMed2000

Rapid diagnosis of acute pyogenic meningitis by a combined PCR dot-blot assay.

A multiplex PCR was employed to amplify unique conserved sequences of DNA from the pathogens Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae from cerebrospinal fluid samples of patients suffering from acute pyogenic meningitis. The accurate identification of the PCR amplified product was achieved by hybridizing dot-blots of the PCR products to probes which were specific, biotinylated internal sequences of the amplified target DNA. Detection of the hybrids was done in a colour reaction using streptavidin-alkaline phosphatase conjugate and BCIP/NBT substrates. The entire protocol took only 7 h for the correct identification of the pathogen present in clinical samples of cerebrospinal fluid. The sensitivity and specificity were >95%.

Biotin↗

PubMed2005

Development of a multiplex nested consensus PCR for detection and identification of major human herpesviruses in CNS infections.

BACKGROUND: Rapid, sensitive and economical detection and identification of human herpesviruses as causative agents of central nervous system (CNS) infections are of clinical importance. The traditional methods for the detection of herpesviruses in CNS infections all suffer from limitations. PCR has a potential to overcome each of them. OBJECTIVES: The aims of this study were reducing the number of primers in multiplex PCR and increasing the sensitivity of the assay by nested PCR. STUDY DESIGN: A multiplex nested consensus PCR (MNC-PCR) was developed for the simultaneous detection of major human herpesviruses. A pair of conserved primers was designed for detection of HSV-1, HSV-2, CMV and EBV and another pair of conserved primers for nested PCR. For VZV, a different pair of primers was designed and another pair of primers for nested PCR. A reduction in the number of designed primer pairs (from five pairs to two in both stages of PCR) is an advantage in this assay. One hundred forty-seven cerebral spinal fluid (CSF) samples from patients that showed clinical manifestation of CNS infections were tested. Results of MNC-PCR in CSF samples were compared with those of single PCR assay for each individual DNA virus. Sensitivity of the assay was determined with a plasmid containing VZV DNA binding protein gene and another plasmid for HSV-1 DNA polymerase gene. False negative results (due to the presence of inhibitor of DNA amplification in CSF samples) were avoided by the inclusion of beta2-microglobulin primers in the MNC-PCR assay as an internal control. RESULTS: Positive results were obtained in 20 CSF samples (8 HSV-1, 2 HSV-2, 4 CMV, 3 VZV, 3 HSV-1/CMV, CMV/VZV and HSV-1/EBV coinfections). The comparison between single PCR and MNC-PCR showed a marked increase in sensitivity of MNC-PCR test, since six negative samples in single PCR proved positive in MNC-PCR (P<0.005). Sensitivity was determined 1-5 plasmid copies for VZV and 50-100 plasmid copies for HSV-1. CONCLUSIONS: The MNC-PCR assay presented in this study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of CSF samples.

Adult↗

PubMed1999

Rapid and precise genotyping of porcine microsatellites.

Microsatellites are useful markers for genetic mapping and linkage analysis because they are highly polymorphic, abundant in genomes and relatively easily scored with polymerase chain reaction (PCR). A rapid genotyping system for microsatellites was developed, which included multiplex PCRs, multiple use of Hydrolink gels, automated fluorescent detection of fragments on an A.L.F. DNA sequencer, automatic assignment of alleles to each locus and verification of genotypes with a self-developed computer program "Fragtest". Eight multiplex PCRs have been developed to genotype 29 microsatellites for genetic and quantitative trait loci (QTL) mapping on pig chromosomes 6, 7, 12 and 13. Three to six microsatellites could be amplified in one multiplex PCR. Each multiplex reaction required only different concentrations of each pair of primers and a low concentration of dNTP (100 microM). A dNTP concentration of 100 microM proved to be optimal for the coamplification of microsatellites under the concentration of 1.5 mM MgCl2. Using four internal size standards added in each sample, the 5% Hydrolink gel could subsequently be used up to five times (total running time of 500 min) on the A.L.F. automated sequencer without significant loss of resolution and precision of fragment length analysis. Automatic assignment of alleles on each locus using "Fragtest" significantly increased the efficiency and precision of the genotyping. This system is thus a rapid, cheap, and highly discriminating genotyping system.

Animals↗

PubMed1995

Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria.2+ fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid.

Bacteria↗

PubMed2006

Changing pattern of human group A rotaviruses: emergence of G12 as an important pathogen among children in eastern India.

BACKGROUND: Rotavirus genotypes, G1-G4 and G9 are associated with childhood diarrhoea throughout the world. In our previous study, we detected G1, G2, G4 and three G12 strains from Kolkata, India. OBJECTIVES: To study the prevalence of G- and P-genotypes of rotaviruses associated with dehydrating diarrhoea in children admitted to two leading hospitals in eastern India. STUDY DESIGN: An active surveillance was conducted for elucidation of rotavirus infection in two leading hospitals in Kolkata, West Bengal and Berhampur (GM), Orissa, India, separated by 603km from January 2003 to April 2005. The rotaviruses were detected by RNA electrophoresis in polyacrylamide gels. G- and P-typing of the positive samples were accomplished by amplifying VP7 and VP4 genes by RT-PCR and genotyped by seminested multiplex PCR methods. Sequencing, sequence analysis and phylogenetic analysis of VP7 genes of G12 strains were carried out to understand the variations between the strains isolated from different parts of the world. RESULTS: The genotypic distribution varied remarkably from our earlier study period (1998-2001) with G1 (53.8%) being the most predominant strain followed by G2 (22.5%), G12 (17.1%), G9 (2.1%) and not a single G3 or G4 isolate was detected separately. 35.2% samples exhibited mixed P-types followed by P[4] (31.7%), P[8] (21.8%) and P[6] (9.8%). The phylogenetic analysis of G12 strains revealed that the G12 strains detected from different parts of the world clustered into three different lineages. Though VP7 sequences of G12 strains isolated from Kolkata and Berhampur are conserved, their P-types were different. CONCLUSION: During this study period we reported emergence of G12 strains as an important pathogen among children in eastern India, thus necessitating its inclusion in future polyvalent vaccine to control rotavirus diarrhoea.

Antigens, Viral↗

PubMed2006

Differential molecular identification of Taeniid spp. and Sarcocystis spp. cysts isolated from infected pigs and cattle.

In the present work, the species-specific identification of Taeniid spp. cysticerci and sarcocystis cysts isolated from infected pigs and cattle was achieved by PCR. In particular: (i) multiplex-PCR derived from HDP2 DNA fragment, specific for Taenia saginata/Taenia solium; (ii) PCRs and PCR-RFLPs of the rDNA internal transcribed spacers 1 and 2 (ITS1 and ITS2) for the differential diagnosis of taeniids; (iii) PCR derived from the 18S rRNA gene and sequencing, specific for Sarcoystis spp. The combined application of these three PCR protocols provided an unequivocally specific diagnosis of T. saginata, T. solium, T. hydatigena, Sarcocystis hominis and Sarcocystis suihominis, and may have practical application in the identification of calcified degenerating or morphologically dubious cysts, for example in the slaughter house situation or in human biopsy samples.

Animals↗

PubMed2006

A two-step protocol for the detection of rearrangements at the AZFc region on the human Y chromosome.

The AZFc region on the human Y chromosome consists mainly of very long direct and inverted repeats and is prone to rearrangement. Although deletion of the entire AZFc is found only in subfertile men, duplications and deletions of portions of AZFc as well as inversions are quite common and represent major polymorphisms of the Y chromosome. Several methods are available to detect these rearrangements, and each has its own advantages and limits. We designed a two-step PCR protocol to study the polymorphic structure of AZFc. The first PCR determines the copy number of the Deleted in Azoospermia (DAZ) genes within AZFc using the autosomal DAZ-Like gene as a dosage control, and the results could be verified by dosage Southern blot analyses. The second PCR simultaneously detects five sequence tagged sites (STSs) that are either present or absent in the various AZFc partial deletions. One of the STSs, sY1291, was found to be polymorphic in size due to varying lengths of a poly-T stretch. A combination of the DAZ dosage PCR and the 5-STS multiplex PCR reaction detects most, if not all, deletions and duplications at AZFc. It offers a simple and reliable way to screen large populations for AZFc rearrangements and study their effects on male fertility.

Blotting, Southern↗

PubMed2001

The detection of Shiga toxin-producing Escherichia coli in diagnostic bovine faecal samples using vancomycin-cefixime-cefsulodin blood agar and PCR.

The prevalence of complex Shiga toxin-producing Escherichia coli (STEC), i.e. STEC containing accessory virulence factors intimin (eaeA) and/or enterohaemorrhagic E. coli haemolysin (ehxA) and their serotypes were determined in diagnostic bovine faecal samples processed during a 3 months period. The presence of complex STEC was determined using PCR and vancomycin-cefixime-cefsulodin blood agar (BVCCA) using a dual approach which involved (i) direct culture of faecal samples on BVCCA followed by mutiplex PCR of BVCCA positive colonies and (ii) culture of faecal samples enriched in modified EC (mEC) broth (with a complex STEC profile determined by PCR) on BVCCA followed by multiplex PCR of BVCCA positive colonies. Using both techniques complex STEC were isolated from 23 (18.7%) of the 123 faecal samples. Complex STEC were isolated from 14 faecal samples by direct culture on BVCCA and 13 faecal samples yielded complex STEC by culture of mEC broths with a complex STEC profile on BVCCA. Only four samples were positive using both techniques. The serotypes isolated included O5:H-, O26:H-, O26:H11, O91:H21, O111:H-, O111:H8, O104:H11, O113:H21 and O157:H8. This study confirms that non-O157 STEC can be isolated from bovine faeces and that they carry types associated with human disease. This work also demonstrates that the use of a dual approach is advisable to increase the likelihood of isolating complex STEC.

Adhesins, Bacterial↗

PubMedDate not supplied

[Population genetics of the 10 short tandem repeat (STR) loci in the population of Podlasie (Northeastern Poland)].

A population study on 10 STR loci: D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01 and FGA was performed on 620 unrelated individuals born in Podlasie (NE Poland). The DNA was amplified by multiplex PCR using a commercially available multiplex AmpFISTR SGM Plus kit. Capillary electrophoresis with fluorescent detection was carried out using the ABI 310 Genetic Analyzer. The expected performance of the analysed loci for personal identification and paternity testing was estimated. All loci met the Hardy-Weinberg assumption. No significant differences between the studied population and other Polish data were found. The combined values of the Matching Probability and of the Power of Exclusion are 4.18 x 10(-13) and 0.99997, respectively.

Adult↗

PubMed2005

Severe hemolytic anemia in a Vietnamese family, associated with novel mutations in the gene encoding for pyruvate kinase.

BACKGROUND AND OBJECTIVES: Chronic hemolytic anemias are very frequent diseases in intertropical countries mainly caused by hemoglobin disorders. We studied a Vietnamese family in which a first child suffered from a severe transfusion-dependent anemia. The family requested an antenatal diagnosis during a second pregnancy. To characterize the molecular defect, we studied the family over three generations. DESIGN AND METHODS: Blood from family members was sampled for a full hematologic evaluation, including enzymatic dosage, and DNA analysis was performed for patients displaying pyruvate kinase deficiency (PK-R). Mutation research on the 11 exons of the PKLR gene was done using a scanning method and sequencing. Deletion was evidenced by a Sybergreen based quantitative real time polymerase chain reaction (PCR) and mapped using quantitative multiplex PCR of short fluorescent fragments spread along the whole sequence of the PKLR gene. RESULTS: Hematologic and molecular studies of this severe chronic anemia demonstrated the existence of two defects in the PKLR gene, a new mutation located on exon 7: c.948C->G (N316K) and a large deletion extending from exon 4 to exon 10. INTERPRETATION AND CONCLUSIONS: We describe a family in a south-east Asian country; the proband had severe transfusion-dependent chronic anemia caused by the association between two PKLR gene mutations, PK Saigon (N316K) and PK Viet del 4-10. Severe chronic anemia could be induced by various molecular defects mainly affecting the globin genes. However, even in populations in which hemoglobin diseases are frequent, enzymatic diseases should be considered.

Adult↗

PubMed1997

p53 alteration and human papilloma virus infection in paranasal sinus cancer.

BACKGROUND: Inverted papilloma (IP) of the paranasal sinus is a benign neoplastic condition that can be associated with squamous cell carcinoma (SCC). To understand the etiology of the disease better, paranasal sinus tumor specimens were examined for alterations in either p53 protein expression or genomic DNA sequence, and for infection by human papilloma virus (HPV). METHODS: Tumor specimens were categorized as follows: benign, nondysplastic IP; IP with dysplasia; SCC arising within IP; or SCC without IP. Sections of each tumor specimen were stained for p53 protein overexpression, and mutations in exons 5-9 of the p53 gene were determined in DNA purified from all tumor samples. HPV infection was screened by degenerate polymerase chain reaction (PCR) amplification and typed by multiplex PCR and direct DNA sequencing of PCR-amplified HPV sequences. RESULTS: Altered p53, either in genetic sequence or protein overexpression, was observed in 0 of 7 benign, nondysplastic IP specimens. A significantly higher p53 alteration incidence was observed for IP specimens exhibiting dysplasia (57%; P < 0.05) and IP specimens that were associated with SCC (75%; P < 0.025). HPV sequences were detected in 9 of 24 (38%) tumor specimens, 78% of which were of the oncogenic HPV16 strain. A significantly higher incidence (P < 0.05) of HPV infection was observed in IP tumors exhibiting dysplasia or containing SCC than in nondysplastic IPs. None of the p53-mutated tumors were infected with oncogenic HPV16. CONCLUSIONS: These data suggest that p53 alterations and/or HPV infection are associated predominantly with IPs exhibiting evidence of dysplasia or IPs associated with SCC, but not in nondysplastic, benign IPs. In addition, an inverse correlation may exist between oncogenic HPV infection and p53 alterations in paranasal sinus tumors. The authors postulate that patients with IPs containing altered p53 may be at increased risk for SCC of the paranasal sinus.

DNA Mutational Analysis↗

PubMed2002

Genetic follow-up of male offspring born by ICSI, using a multiplex fluorescent PCR-based test for Yq deletions.

De-novo deletions involving AZFa, b, c and d are one of the most common chromosomal aberrations in man resulting in defective spermatogenesis and male infertility. Currently, Yq deletion screening involves either single or multiplex PCR using Y-specific sequence tagged site markers and the subsequent analysis of the amplification products on ethidium bromide-stained agarose gels. To improve the practicality of routine and high throughput Yq testing, we have developed a more sensitive multiplex fluorescent (FL)-PCR screening system using genomic DNA extracted from cheek buccal cells as a readily available PCR template. For genetic follow-up studies of ICSI-conceived children, we also developed a DNA fingerprinting system based on the co-amplification of four highly polymorphic markers to validate family samples and detect any potential extraneous DNA contamination that could cause a misdiagnosis. Multiplex FL-PCR analysis of buccal cell DNA from two infertile men who conceived three sons by ICSI demonstrated that their Yq deletions were vertically transmitted. Fine mapping with additional Yq markers revealed identical deletion endpoints involving the loss of AZFdc sequences. This firstly indicates that the extent of the Yq deletion was unchanged on ICSI transmission and secondly supports the view that AZFdc deletions may arise by a common de-novo event. Analysis of paternal, maternal and sibling DNA fingerprints showed the co-inheritance of parental alleles by each male child and confirmed the expected relationship between each family member. The application of these new FL-PCR based screening tests in genetic follow-up studies will assist in confirming transmission of specific genetic defects to male offspring conceived by ICSI and provide a basis for genetic counselling and potential treatment options as these boys approach sexual maturity.

Adult↗

PubMed2006

[Detection of fusion genes associated with specific translocations in acute leukemia patients with normal karyotypes by using multiplex RT-PCR].

This study was aimed to explore the usefulness of multiplex reverse transcription-polymerase chain reaction (multiplex RT-PCR) in detection of fusion genes associated with specific translocations in acute leukemia (AL) patients with normal karyotypes. 37 AL patients with normal karyotypes were analyzed by multiplex RT-PCR. The results showed that 4 types of fusion genes such as PML/RARA, AML1/ETO, CBFbeta/MYH11, BCR/ABL were detected in 8 (21.6%) patients by multiplex RT-PCR. In conclusion, multiplex RT-PCR is useful in detection of fusion genes associated with specific translocations in acute leukemia (AL) with normal karyotypes and it would refine the karyotype analysis. When the normal karyotypes were detected in acute leukemia patients by conventional cytogenetic method, the multiplex RT-PCR should be performed for them.

Acute Disease↗

PubMed2002

Detection of enteroviruses and hepatitis A virus in water by consensus primer multiplex RT-PCR.

AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-,671-, 1084-, and 1128bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus,21 PFU for Coxsackie virus,60 PFU for Echovirus and 105 TCID(50) for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID(50) for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness.

Base Sequence↗

PubMed2000

Use of two reporter dyes without interference in a single-tube rapid-cycle PCR: alpha(1)-antitrypsin genotyping by multiplex real-time fluorescence PCR with the LightCycler.

BACKGROUND: alpha(1)-Antitrypsin is the major plasma serine protease inhibitor. Its deficiency is mainly associated with the alleles PI*S and PI*Z and can lead to obstructive lung disease in adults and to liver cirrhosis during childhood. METHODS: A multiplex PCR method has been established that uses two sets of primers to amplify the gene regions covering the PI*S or PI*Z mutations sites. Mutation detection was performed on the LightCycler by melting curve analysis of detection probes labeled with two different fluorescent dyes, LC-Red640 and LC-Red705. RESULTS: Unequivocal genotyping results were obtained for all investigated samples in an assay time of approximately 30 min. The color compensation procedure greatly improved the readability of the resulting diagnostic melting curves. CONCLUSIONS: To our knowledge, this is the first report of simultaneous detection of two mutations in a single tube by PCR of genomic DNA and the use of two different reporter dyes with the LightCycler color compensation feature. This approach is a rapid, convenient, and economic alternative to other methods described to date for the detection of alpha(1)-antitrypsin deficiency alleles.

DNA↗

PubMedDate not supplied

Experimental evidence of indirect transmission of Mycoplasma synoviae.

The aim of the study was to analyse experimental transmission of Mycoplasma synoviae, an avian pathogen. Three experiments using specific pathogen-free day-old chicks placed in isolators were conducted. In the first experiment, the birds were introduced in an isolator previously contaminated with a M. synoviae broth culture. After 34 days, these birds were eliminated and, for the second trial, the chicks were introduced in the same isolator without disinfecting. In the third assay, the chicks were placed in an isolator containing a mixture of food, feathers and dust collected less than an hour earlier from a M. synoviae infected laying hen flock. In the second and third experiments in order to exacerbate the M. synoviae infection, the birds were inoculated with infectious bronchitis (IB) virus. The presence of M. synoviae in the environment and in tracheal swabs was monitored by culture, a multiplex PCR (mPCR) detecting M. synoviae and Mycoplasma 16S rDNA and a multiplex RT-PCR (mRT-PCR) detecting the M. synoviae mRNA coding for a membrane protein and Mycoplasma 16S rRNA. In in vitro experimental conditions, M. synoviae mRNA and 16S rRNA were detected up to 20 min and 23 h respectively after mycoplasma death. In the first assay, the first infected bird was detected on the 13th day. In the second trial, culturable M. synoviae or viable M. synoviae were detected in the isolator for 3 or 4 to 5 days respectively after depopulation of the birds of the first assay whereas the first culture positive tracheal swabs were detected on the 33rd day, after IB inoculation. In the third experiment, the first infected birds were detected on the 54th day. Thus, the different assays showed that M. synoviae contaminated material (dust, feathers and food) can infect chicks, sometimes after remarkably long silent periods.

Animals↗

PubMed2006

Prevalence of Campylobacter species, Helicobacter pylori and Arcobacter species in stool samples from the Venda region, Limpopo, South Africa: studies using molecular diagnostic methods.

OBJECTIVES: This study determined the prevalence of Campylobacter spp., Helicobacter pylori and Arcobacter spp. in stool samples from Venda in relation to diarrhea, intestinal inflammation and HIV status using specific molecular methods. METHODS: Stool samples were collected from hospital patients (255) and primary school children (67). Genomic DNA was extracted from the stools and molecular methods including PCR, PCR followed by restriction analysis and multiplex PCR were used to test for the different organisms. The lactoferrin content of the stools was determined using commercial kits from TechLab (Blacksburg, VA, USA). RESULTS: The prevalence of the different organisms was 50.6% for H. pylori, 10.2% for C. jejuni, 6.2% for A. butzleri, 6.5% for C. coli, 3.1% for C. concisus, 2.8% for A. cryaerophilus and 1.9% for A. skirrowii. Of all the organisms, only C. jejuni was significantly associated with diarrhea (84.8%) (chi2=21.025, P<0.001) and elevated levels of lactoferrin (78.8%) (chi2=16.919, P<0.005) and was an important pathogen associated with diarrhea among HIV positive individuals (22.8%). CONCLUSIONS: Campylobacter infections are common causes of gastroenteritis in Venda. Non-C. jejuni/coli Campylobacters such as C. concisus as well as A. butzleri and H. pylori may be involved in gastrointestinal diseases in the region but further studies are needed to confirm this hypothesis.

Adolescent↗

PubMed1994

Preimplantation diagnosis of cystic fibrosis by simultaneous detection of the W1282X and delta F508 mutations.

W1282X (W) and delta F508 (delta) are the two most common mutations of the cystic fibrosis Israeli population. Patients who are homozygotes (WW and delta delta) as well as compound heterozygotes (W delta) present a severe phenotype of the disease. In the present study, we have developed a polymerase chain reaction (PCR)-based method for the detection of both mutations simultaneously in a single blastomere. Unfertilized human oocytes and single polyspermic blastomeres were subjected to a two-round PCR amplification: a first round of multiplex PCR followed by a second round of nested PCR, done separately at each locus. Clear signals at both loci were obtained in 51% (47/65) of oocytes and 69% (24/35) of blastomeres. The genotype of the single cell analysed was determined by endonuclease digestion of the W products and by heteroduplex formation of the delta F products. This diagnostic system will allow the identification of affected embryos (WW, delta delta, W delta) as well as phenotypically normal carriers (W+, +delta), and therefore may be used for cystic fibrosis preimplantation diagnosis in families who carry either or both mutations.

Base Sequence↗