OBJECTIVES: To develop a multiplex PCR amplification system (EX20+30Y for short) of 19 autosomes, 30 Y-STR loci plus the gender indicator, and evaluate its forensic application value. METHODS: With the six-color fluorescence labeling technology, a multiplex amplification system of 19 autosomal STR loci and 30 Y-STR loci plus the gender indicator was constructed. Blood samples from 210 unrelated individuals, 69 daily case samples and standard samples 9948 and 9947A were collected for loci detection and analysis. The EX20+30Y multiplex amplification system was evaluated by its sensitivity, mixed sample detection ability, species specificity, balance, direct amplification ability, sample applicability and anti-inhibition ability. RESULTS: Multiplex amplification of blood samples from 210 unrelated individuals by the detection system obtained accurate genotyping results. The detection sensitivity of standard samples was 0.125 ng and the species specificity was high. The 69 samples from daily cases were genotyped correctly. Moreover, standard sample 9948 could be accurately genotyped even if the samples contained a certain concentration of inhibitors. CONCLUSIONS The multiplex amplification system established in this study can conduct combined examination of 19 autosomes, 30 Y-STR loci plus the gender indicator with accurate genotyping and high sensitivity. It has a good forensic application prospect.
Chromosomes, Human, Y/genetics* ; DNA Fingerprinting/methods* ; Forensic Medicine/methods* ; Humans ; Microsatellite Repeats ; Multiplex Polymerase Chain Reaction ; Species Specificity

A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.
Animals ; Bacteria ; genetics ; isolation & purification ; Bacteriological Techniques ; methods ; Endometritis ; microbiology ; veterinary ; Female ; Multiplex Polymerase Chain Reaction ; standards ; Polymerase Chain Reaction ; veterinary ; Sensitivity and Specificity ; Sheep ; Sheep Diseases ; microbiology ; Tibet

BACKGROUND: Mycobacterium bovis Bacille Calmette-Guérin (BCG) osteitis, a rare complication of BCG vaccination, has not been well investigated in Korea. This study aimed to evaluate the clinical characteristics of BCG osteitis during the recent 10 years in Korea. METHODS: Children diagnosed with BCG osteitis at the Seoul National University Children's Hospital from January 2007 to March 2018 were included. M. bovis BCG was confirmed by multiplex polymerase chain reaction (PCR) in the affected bone. BCG immunization status and clinical information were reviewed retrospectively. RESULTS: Twenty-one patients were diagnosed with BCG osteitis and their median symptom onset from BCG vaccination was 13.8 months (range, 6.0–32.5). Sixteen children (76.2%) received Tokyo-172 vaccine by percutaneous multiple puncture method, while four (19.0%) and one (4.8%) received intradermal Tokyo-172 and Danish strain, respectively. Common presenting symptoms were swelling (76.2%), limited movement of the affected site (63.2%), and pain (61.9%) while fever was only accompanied in 19.0%. Femur (33.3%) and the tarsal bones (23.8%) were the most frequently involved sites; and demarcated osteolytic lesions (63.1%) and cortical breakages (42.1%) were observed on plain radiographs. Surgical drainage was performed in 90.5%, and 33.3% of them required repeated surgical interventions due to persistent symptoms. Antituberculosis medications were administered for a median duration of 12 months (range, 12–31). Most patients recovered without evident sequelae. CONCLUSION: Highly suspecting BCG osteitis based on clinical manifestations is important for prompt management. A comprehensive national surveillance system is needed to understand the exact incidence of serious adverse reactions following BCG vaccination and establish safe vaccination policy in Korea.
Child ; Drainage ; Femur ; Fever ; Humans ; Immunization* ; Incidence ; Korea* ; Methods ; Multiplex Polymerase Chain Reaction ; Mycobacterium bovis* ; Mycobacterium* ; Osteitis* ; Punctures ; Retrospective Studies ; Seoul ; Tarsal Bones ; Vaccination

Enterococcus spp. are opportunistic pathogens that cause lameness in broiler chickens, resulting in serious economic losses worldwide. Virulence of Enterococcus spp. is associated with several putative virulence genes including fsr, efm, esp, cylA, cad1, ace, gelE, and asa1. In this study, multiplex polymerase chain reaction (PCR) for the simultaneous detection of these virulence genes in Enterococcus spp. was developed, and detection limits for E. faecium, E. faecalis, and E. hirae were 64.0 pg/µL, 320.0 pg/µL, and 1.6 ng/µL DNA, respectively. Among 80 Enterococcus isolates tested, efm and cad1 were detected in all 26 E. faecium samples, and only cad1 was observed in E. hirae. Additionally, the presence of virulence genes in 25 E. faecalis isolates were 100% for cad1, 88.0% for gelE, 64.0% for fsr, 44.0% for asa1, 16.0% for cylA, and 4.0% for esp. No virulence genes were found in E. gallinarum isolates. A total of 49 isolates were resistant to tigecycline and to at least 2 different classes of antibiotics. The most prevalent resistance was to ciprofloxacin (73.5%), quinupristin/dalfopristin (55.1%), and tetracycline (49.0%). No strains were resistant to vancomycin or linezolid. This is the first multiplex PCR assay to simultaneously detect eight virulence genes in Enterococcus spp., and the method provides diagnostic value for accurate, rapid, and convenient detection of virulence genes. Additionally, we report the prevalence of virulence genes and antimicrobial resistance in Enterococcus isolates from commercial broiler chickens suffering lameness.
Anti-Bacterial Agents ; Chickens ; Ciprofloxacin ; DNA ; Drug Resistance, Microbial ; Enterococcus ; Limit of Detection ; Linezolid ; Methods ; Multiplex Polymerase Chain Reaction ; Prevalence ; Tetracycline ; Vancomycin ; Virulence

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.
Bacteriophage T4 ; Cryptosporidium parvum ; Cryptosporidium ; Cyclospora ; Diagnosis ; Diarrhea ; DNA ; Fluorescent Dyes ; Giardia lamblia ; Giardia ; Glutamate Dehydrogenase ; Humans ; Limit of Detection ; Methods ; Multiplex Polymerase Chain Reaction ; Oocysts ; Parasites ; Real-Time Polymerase Chain Reaction

BACKGROUND: The aim of this study was to investigate the status of BRCA1/2 genetic testing practices in Korea in 2014. METHODS: A structured questionnaire was provided to the specialist in charge of BRCA1/2 genetic testing via e-mail between 28 July and 10 August 2015. A total of 11 genetic testing professionals from 14 organizations responded to the survey that asked about the status of BRCA1/2 genetic testing in the year 2014. RESULTS: The average number of BRCA1/2 genetic tests executed was 192; 6 organizations had executed less than 100 tests, and 5 organizations had conducted more than 100 tests. The primary testing method used was Sanger sequencing (100%), and 2 institutes performed multiplex ligation-dependent probe amplification (MLPA). The analysis software differed across the various organizations, with Sequencher (81.81%), Seqscape (27.27%), and Codoncode Aligner (9.09%) reported as utilized. We found that the guidelines for the interpretation of the genetic tests were different at each institution. CONCLUSIONS: Although this study only examined the status of the 2014 BRCA1/2 genetic testing practices of 11 institutions, it illustrates the necessity for standardized genetic testing or interpretation guidelines in Korea.
Academies and Institutes ; Electronic Mail ; Genetic Testing* ; Korea* ; Methods ; Multiplex Polymerase Chain Reaction ; Specialization ; Surveys and Questionnaires

OBJECTIVETo explore the value of multiplex ligation-dependent probe amplification (MLPA) for the detection of chromosome abnormalities in miscarriage tissues, and to correlate the result with ultrasound findings.

METHODSA total of 421 cases of spontaneous abortions and fetal deaths were detected with the MLPA method.

RESULTSAmong the 421 samples, 232 (55.11%) had an abnormal MLPA result. For the 286 cases derived from < 13 weeks pregnancy, 206 (72.03%) were abnormal. For the 49 cases from 14-19 weeks pregnancy, 14 (28.57%) were abnormal. For the 86 cases derived after 20 weeks pregnancy, 12 (13.95%) were abnormal. Among the 117 cases with abnormal ultrasound findings, 33 cases (28.21%) had an abnormal MLPA result, 28 out of the 33 cases were numerical chromosome abnormality, 4 cases were chromosome microdeletion and/or micro duplication, 1 case had both numerical abnormality and microduplication. For those with abnormal ultrasound findings for the neck region, fetal edematous syndrome, multiple malformations and digestive system, the detection rates for MLPA were 71.4%, 58.8%, 37.8%, and 9.1%, respectively. For those with abnormal finding of cardiac system, nervous system, face, skeletal system and urinary system, none was found with positive results of MLPA.

CONCLUSIONNumerical chromosomal abnormalities account for the majority of cases with spontaneous abortion. With the increase of gestational age, the occurrence of chromosomal abnormalities gradually declines. Combined ultrasound and MLPA assay can improve the detection rate and accuracy for chromosomal abormalities.

Abortion, Spontaneous ; diagnostic imaging ; genetics ; Chromosome Deletion ; Chromosome Disorders ; diagnostic imaging ; genetics ; Chromosome Duplication ; DNA ; analysis ; Female ; Fetal Diseases ; diagnostic imaging ; genetics ; Gestational Age ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Pregnancy ; Reproducibility of Results ; Sensitivity and Specificity ; Telomere ; genetics ; Ultrasonography, Prenatal ; methods

OBJECTIVETo assess the value of G-banded karyotyping in combination with multiplex ligation-dependent probe amplification (MLPA) as a tool for the detection of chromosomal abnormalities in fetuses with congenital heart defects.

METHODSThe combined method was used to analyze 104 fetuses with heart malformations identified by ultrasonography. Abnormal findings were confirmed with chromosomal microarray analysis (CMA).

RESULTSNineteen (18%) fetuses were found to harbor chromosomal aberrations by G-banded karyotyping and MLPA. For 93 cases, CMA has detected abnormalities in 14 cases including 10 pathogenic copy number variations (CNVs) and 4 CNVs of uncertain significance (VOUS). MLPA was able to detect all of the pathogenic CNVs and 1 VOUS CNV.

CONCLUSIONCombined use of G-banded karyotyping and MLPA is a rapid, low-cost and effective method to detect chromosomal abnormalities in fetuses with various heart malformations.

Chromosome Aberrations ; Chromosome Banding ; Chromosome Disorders ; diagnosis ; genetics ; DNA Copy Number Variations ; Female ; Fetal Diseases ; diagnosis ; genetics ; Genetic Testing ; methods ; Heart Defects, Congenital ; diagnosis ; genetics ; Humans ; Karyotyping ; methods ; Multiplex Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; methods ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVELower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes.

METHODSThree multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens.

RESULTSThe detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens.

CONCLUSIONThis study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.

Bacteria ; classification ; genetics ; isolation & purification ; Bacteriological Techniques ; DNA, Bacterial ; genetics ; Electrophoresis, Capillary ; methods ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Respiratory Tract Infections ; microbiology ; Sensitivity and Specificity

OBJECTIVES: Studies on Clostridium difficile are rare in Korea. We investigated the epidemiological characteristics of C. difficile isolates from patients with C. difficile-associated disease (CDAD) in Korea. METHODS: Multiplex polymerase chain reaction was performed to detect the presence of tcdA and tcdB toxin genes. Antimicrobial susceptibility test was carried out by the disk-dilution method. C. difficile strains were subtyped by automated repetitive-element palindromic PCR (rep-PCR). RESULTS: Among patients with CDAD, 73 (25.8%), 32 (11.3%), 32 (11.3%), and 26 (9.2%) suffered from pneumonia, cancer or neoplasm, diabetes, and colitis, respectively. Of all stool samples, 43 samples (15.2%) were positive for C. difficile strains. We observed two expression patterns of toxin genes: tcdA+/tcdB+ (86% isolates) and tcdA−/tcdB+ (14% isolates), with all isolates expressing tcdB. Furthermore, some isolates were resistant to clindamycin (65%), ampicillin (56%), and cefazolin (40%), but all were susceptible to vancomycin and metronidazole. The tested samples were classified into diverse clusters using automated rep-PCR. CONCLUSION: Our findings revealed the characteristics and antibiotic resistance of C. difficile isolates from patients in Korea. The epidemiological data may provide valuable insight into development of treatment strategies for C. difficile infections in Korea.
Ampicillin ; Cefazolin ; Clindamycin ; Clostridium difficile* ; Clostridium* ; Colitis ; Drug Resistance, Microbial ; Humans ; Korea* ; Methods ; Metronidazole ; Multiplex Polymerase Chain Reaction ; Pneumonia ; Polymerase Chain Reaction ; Vancomycin