Multiplex Polymerase Chain Reaction System Research Articles

Development of a multiplex microsatellite assay for parentage assignment in orange‐spotted groupers (Epinephelus coioides)

AbstractEstablishment of reliable pedigree information of different lineages is crucial for selective breeding and stock enhancement programs. However, owing to the absence of a reliable assay for parentage assignment, such information is lacking in orange‐spotted grouper (Epinephelus coioides), which is an economically important coral‐reef fish. In this study, we developed two multiplex microsatellite polymerase chain reaction systems that consisted of 12 microsatellite markers (seven in system A and five in system B) for parentage assignment of orange‐spotted groupers. The theoretical exclusion probability per locus ranged from 0.079 to 0.436 when both parents were unknown (E‐1P) and from 0.191 to 0.614 when one parent was known (E‐2P). The combined exclusion probability values of 12 loci were 98.8% (E‐1P) and 100% (E‐2P), respectively. The accuracy of the parentage test for a mixed family (15 dams and 12 sires, 226 offspring) was 100%. In addition, two wild populations (79 individuals) were added as interferential offspring, only four of which were misassigned to the stock. The success rate of assignment was 98.69%. We provide a reliable microsatellite‐based assay for parentage assignment in orange‐spotted groupers, which will be useful for selective breeding and stock enhancement programs for this species and maybe other groupers.

Development of a multiplex PCR system to discriminate the alleles of the brown planthopper resistance locus BPH1 in rice (Oryza sativa L.)

The brown planthopper, Nilaparvata lugens Stal (Hemiptera: Delphacidae), is a serious insect pest of rice (Oryza sativa L.). It is known that several wild rice and some Indica Group rice cultivars carry brown planthopper resistance genes. The brown planthopper resistance locus BPH1 has been used for the development of brown planthopper resistance rice in Southeast Asia. Bph1 and Bph2 are considered to be different resistance alleles in the same BPH1 locus, and each resistance allele has a different specificity against different brown planthopper biotypes. The first and second intronic DNA sequences in the BPH1 locus, which are derived from the Indica Group rice cultivar ADR52, wild rice Oryza officinalis and the Japonica Group rice cultivar Nipponbare, were analyzed to find insertion and deletion (InDel) polymorphisms. Based on this InDel polymorphisms information, we developed a multiplex polymerase chain reaction (PCR) method that enables the simultaneous identification of three alleles from the BPH1 locus: Bph1, Bph2 and a Japonica-type allele. Our method will be useful for future breeding research because it facilitates the classification of allele types in the BPH1 locus.

Development and application of perfect SSR markers in cotton

BackgroundThis study aimed to develop a set of perfect simple sequence repeat (SSR) markers with a single copy in the cotton genome, to construct a DNA fingerprint database suitable for authentication of cotton cultivars. We optimized the polymerase chain reaction (PCR) system for multi-platform compatibility and improving detection efficiency. Based on the reference genome of upland cotton and 10× resequencing data of 48 basic cotton germplasm lines, single-copy polymorphic SSR sites were identified and developed as diploidization SSR markers. The SSR markers were detected by denaturing polyacrylamide gel electrophoresis (PAGE) for initial screening, then fluorescence capillary electrophoresis for secondary screening. The final perfect SSR markers were evaluated and verified using 210 lines from different sources among Chinese cotton regional trials.ResultsUsing bioinformatics techniques, 1 246 SSR markers were designed from 26 626 single-copy SSR loci. Adopting a stepwise (primary and secondary) screening strategy, a set of 60 perfect SSR markers was selected with high amplification efficiency and stability, easy interpretation of peak type, multiple allelic variations, high polymorphism information content (PIC) value, uniform chromosome distribution, and single-copy characteristics. A multiplex PCR system was established with ten SSR markers using capillary electrophoresis detection.ConclusionsA set of perfect SSR markers of cotton was developed and a high-throughput SSR marker detection system was established. This study lays a foundation for large-scale and standardized construction of a cotton DNA fingerprint database for authentication of cotton varieties.

How reliable is the next generation of multiplex-PCR for diagnosing prosthetic joint infection compared to the MSIS criteria? Still missing the ideal test

Identification of the pathogen in case of a periprosthetic joint infection (PJI) remains 1 of the greatest challenges in septic surgery. Rapid germ identification enables timely, specific, antimicrobial therapy. The first multiplex PCR (polymerase chain reaction) generation (Unyvero-i60) enables germ detection within 5 hours with a sensitivity of 78.8% and a specificity of 100%. The aim of this study is to investigate the performance of the new generation of cartridges (Unyvero-ITI) of multiplex PCR in the case of a PJI. In a prospective study, intraoperatively aspirated synovial fluid from 97 patients with aseptic or septic hip or knee revision surgery (49 aseptic, 48 septic) was examined with the multiplex PCR system (Unyvero-ITI) and the results were compared with the MSIS criteria. In addition, the time until the microbiological result was obtained in the event of a germ detection was documented. The multiplex PCR showed a germ detection with a sensitivity of 85.1% and a specificity of 98.0%. In 7 cases a false negative result was found and in one patient a false positive result was found. The general accuracy of this test procedure was 91.8%. The detection of germs was carried out within 5 hours with the multiplex PCR compared to 4.9 days in conventional microbiological diagnostics. The new generation of multiplex-PCR was able to improve germ detection. The possibility of prompt detection of germs offers the option of faster, targeted antimicrobial therapy. This diagnostic tool offers significant advantages, particularly in the context of an acute periprosthetic infection.

Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products.

Poultry meat is consumed worldwide and is prone to food fraud because of large price differences among meat from different poultry species. Precise and sensitive analytical methods are necessary to control poultry meat products. We chose species–specific sequences of the cytochrome b gene to develop two multiplex real-time polymerase chain reaction (real-time PCR) systems: one for chicken (Gallus gallus), guinea fowl (Numida meleagris), and pheasant (Phasianus colchicus), and one for quail (Coturnix japonica) and turkey (Meleagris gallopavo). For each species, added meat could be detected down to 0.5 % w/w. No cross reactions were seen. For these two real-time PCR systems, we applied three different quantification methods: (A) with relative standard curves, (B) with matrix-specific multiplication factors, and (C) with an internal DNA reference sequence to normalize and to control inhibition. All three quantification methods had reasonable recovery rates from 43% to 173%. Method B had more accepted recovery rates, i.e., in the range 70–130%, namely 83% compared to 75% for method A or C.

Genetic polymorphism analyses of three novel X chromosomal short tandem repeat loci in the Xp22.3 region.

X chromosomal short tandem repeats (X-STRs) can be useful for haplotype analysis in DNA testing, particularly for complex kinship testing or when one parent is absent. We searched downstream of four previously detected loci in the Xp22.3 region (LC149476, LC149479, LC149480, and LC149484) and detected and analyzed three novel short tandem repeats (STRs), LC317283, LC317284, and LC317285, with the repeat sequences TATAA, TTTA, and TATC, respectively. The forensic statistical values in Japanese subjects were confirmed to be noninferior to existing loci, with values for polymorphism information content, the power of discrimination in males (PDm), and the power of discrimination in females (PDf) of 0.5606-0.7448, 0.6078-0.7774, and 0.7990-0.9178, respectively. Haplotype analysis also revealed linkage disequilibrium between LC317283 and the four known loci (LC149476, LC149479, LC149480, and LC149484) and between two other novel loci (LC317284 and LC317285). Analysis of three family samples suggested that these STRs could be useful in complex kinship testing, so we developed an X-STR multiplex polymerase chain reaction (PCR) system for the seven loci and confirmed its ability to provide favorable amplification. We anticipate that the identified loci and developed multiplex PCR system will be beneficial to the field of forensic medicine.

Development of Species-Specific PCR Primers for the Rapid and Simultaneous Identification of the Six Species of Genus Takifugu.

ABSTRACTPufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

Validation of a multiplex amplification system of 19 autosomal STRs and 27 Y-STRs

This article describes a newly devised autosomal short tandem repeat (STR) multiplex polymerase chain reaction (PCR) system for 19 autosomal loci (D12S391, D13S317, D16S539, D18S51, D19S433, D2S1338, D21S11, D3S1358, D5S818, D6S1043, D7S820, D8S1179, CSF1PO, FGA, TH01, TPOX, vWA, Penta D and Penta E), 27 Y-chromosome STR loci (DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS635, DYS627, YGATAH4 and DYF387S1) and amelogenin with six-colour fluorescent labelling. Various parameters were evaluated, such as its accuracy, sensitivity, specificity, stability, ability to analysis of mixtures and effects of changes in the PCR-based procedures. All of the 47 selected STR loci were accurately and robustly amplified from 282 bloodstain samples. The species-specificity was high and some ability to inhibit Hematin was identified. The lowest detectable DNA amount was ≥0.125 ng. All of the male loci of the secondary component were revealed precisely when the control DNA was mixed at male/female and male/male ratios of 1:4 or more. We conclude that the present 19-plex autosomal STR and 27 Y-STR assay is both accurate and sensitive. It constitutes an additional powerful tool for forensic applications.

Chromosomal and Y-chromosome microdeletion analysis in 1,300 infertile males and the fertility outcome of patients with AZFc microdeletions.

The present study investigated the frequency of chromosome aberrations and AZF microdeletions in infertile patients with nonobstructive azoospermia (NOA) or severe oligozoospermia. Additionally, the effect of the AZFc microdeletions on the success of microdissection testicular sperm extraction (microTESE) and intracytoplasmic sperm injection (ICSI) methods were evaluated. Peripheral blood samples were received from 1,300 infertile men with NOA and severe oligozoospermia. Karyotyping and FISH analysis were performed according to standard methods. AZF microdeletions were analysed using multiplex polymerase chain reaction or GML Y-chromosome Microdeletion Detection System consisting of 14 markers. The chromosomal aberrations and the AZF microdeletions frequency among 1,300 infertile men were 10.6% and 4.0% respectively. Either ejaculated spermatozoa or microTESE was performed on only in 19 out of 26 patients with AZFc deletions. Of the 19 patients, four had severe oligozoospermia and 15 had NOA. In eight out of 15 NOA patients, testicular mature spermatozoa were obtained (53.3%) and then ICSI was applied to mature oocytes. After undergoing ICSI treatment, clinical pregnancy and live birth outcome rates were found to be 37.5% and 25% respectively. These results suggest that infertile patients with AZFc microdeletion could achieve successful fertilisation pregnancies with the help of assisted reproductive technology.

643. Comparison of Multiplex Polymerase Chain Reaction (PCR) and Routine Culture for the Detection of Respiratory Pathogens in Pneumonia Patients

BackgroundThe identification of causative pathogens in pneumonia can be challenging, and conventional culture methods can take up to 72 hours. However, rapid microbiologic tests identify organisms within hours. The Biofire®Filmarray ((bioMérieux, North Carolina) Pneumonia Panel was recently approved by the FDA. The multiplex PCR system identifies 33 targets from sputum and bronchoalveolar (BAL) samples, which include 18 bacteria, 8 viruses, and 7 antibiotic resistance genes. The purpose is to compare the panel to routine culture methods for the detection of respiratory pathogens in patients with pneumonia in a 794-bed teaching hospital in northwest Ohio.MethodsWe retrospectively screened all hospitalized intensive care unit patients who met clinical and radiological criteria of pneumonia using electronic medical records between November 2018 and February 2019. Adult patients who had respiratory cultures collected within 7 days were included. Repeat specimens were excluded. Routine cultures were performed using the laboratory’s standard procedure, and Pneumonia Panel testing was performed according to manufacturer instructions.ResultsFifty-nine respiratory or 13 BAL and 46 sputum specimens were evaluated. There was no discrepancy between culture and PCR in 63% (37/59) samples. One (8%) BAL and 10 (22%) sputum specimens had additional pathogens detected by PCR. There was a discrepancy between culture and PCR in four (31%) BAL and seven (15%) sputum samples. The largest discrepancy was noted amongst Serratia marcescens (4/59 or 7%) and Haemophilus influenzae (6/59 or 10%) species. Only one sputum culture had Legionella detected by PCR. Additionally, 17 specimens had a virus detected either alone or with another bacterial pathogen by PCR. For the resistance genes, KPC was detected by PCR but not by Modified Carbapenem Inactivation Method (mCIM) test. The mecA gene was detected in six of seven (86%) of methicillin-resistant Staphylococcus aureus (MRSA) isolates. CTX-M was detected in Serratia and Klebsiella pneunomiae in two samples; however, the organisms were not isolated in culture.ConclusionThe Pneumonia Panel can identify additional bacteria that did not grow in culture. This panel can rapidly identify pathogens and potentially reduce unnecessary antibiotic use.Disclosures All authors: No reported disclosures

High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid malignancies.

Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high-throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high-throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V- and J-segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T-lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.

Profiling of UGT1A1*6, UGT1A1*60, UGT1A1*93, and UGT1A1*28 Polymorphisms in Indonesian Neonates With Hyperbilirubinemia Using Multiplex PCR Sequencing.

Background: Single nucleotide polymorphism (SNP) variants of the uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene have been studied as an important factor in neonatal hyperbilirubinemia (jaundice) severity. Specific ethnicities, including Asians, have certain SNPs that appear more frequently than others.Aim: To identify the most common SNPs in Indonesian neonates and their association with the severity of neonatal hyperbilirubinemia.Methods: Eighty-eight inborn and outborn jaundiced infants from three different hospitals (Bengkulu, Jakarta, Biak Papua) across Indonesia were enrolled in this cross-sectional study and their peak total serum bilirubin (TSB) levels assessed. SNP variant analyses of the TATAA box, promoter, and exon 1 regions of UGT1A1 gene from 78 of the 88 infants were carried out using the SNaPshotR Multiplex Polymerase Chain Reaction (PCR) System followed by DNA sequencing.Results: We detected SNP variants UGT1A1*28, UGT1A1*60, UGT1A1*93, and UGT1A1*6 in our population. Mean total serum bilirubin (TSB) was 14.59 ± 5.57 mg/dL. Bivariate analyses using delivery location, gestational age, birth weight, mother's age, and ethnicity were shown to be associated with moderate-to-severe hyperbilirubinemia (p < 0.05). None of the four SNPs appeared to be associated with moderate-to-severe hyperbilirubinemia. In multivariate analysis, however, only the “other ethnic group” (e.g., Chinese, Bengkulu, Papua, Bima) category showed an association with moderate-to-severe hyperbilirubinemia, with an odds ratio of 6.49 (95% CI 1.01–41.67; p < 0.05).Conclusions: We found that the UGT1A1*60 is the most common SNP detected in neonates with hyperbilirubinemia in the Indonesian population. Interestingly, in Indonesia, UGT1A1 polymorphisms do not appear to be associated with differences in the severity of hyperbilirubinemia.

Multiplex polymerase chain reaction detection of Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected maize leaf tissues

Multiplex polymerase chain reaction (PCR) is a method for simultaneous identification and detection of multiple pathogenic fungi, however, its complexity limits its application. To simplify the protocol and to improve the effectiveness, three-level designs for six factors (three primers, Taq DNA polymerase, dNTP, Mg2+ ) were constructed to optimize the multiplex PCR system by using the orthogonal design method and the annealing temperature of the PCR reactions was also optimized. Finally, a multiplex PCR system for the simultaneous detection of these three pathogens of maize was successfully established. The reaction volume was 25 μl and the annealing temperature was 57℃. The optimal conditions for multiplex PCR reaction contained 0.48 μmol/L Cl-1/Cl-2, 0.72 μmol/L Bm-1/Bm-2, 0.24 μmol/L Az-1/Az-2, 1.5 U polymerase, 0.35 mmol/L dNTP, and 1.25 mmol/L MgCl2 . The multiplex PCR system can detect Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected plant tissues rapidly with the sensitivity at 10 pg DNA/μl.

Iranian patients with hemoglobin H disease: genotype-phenotype correlation.

Thalassemia is one of the most common monogenic hereditary disorders. Despite noticeable advances made in prevention strategies, it is still highly prevalent in the Iranian population. A key approach to management and early diagnosis of the disease is through revealing the regions with high prevalence and determining common genetic and phenotypic diversity. In the current study Hemoglobin H (HbH) disease patients were analyzed as the most common form of thalassemia intermedia in Iran. A total of 80 patients suspected of being thalassemic according to their mild to moderate anemia, microcytosis and normal iron levels were included in this study at the hemoglobinopathy and thalassemia center of Ahvaz University of Medical Science. Patients were analyzed for hematological parameters and HbH mutations using Multiplex Gap Polymerase Chain Reaction and Multiplex Amplification Refractory Mutation System. Twelve mutations were detected in the studied population. The most common genotype was -α3.7/--MED (45%) followed by Homozygote αPoly A2 (17.5%). A total of ten different alpha-globin (α-globin) mutations were observed in patients which --MED, being the most common mutation (26.27%), followed by -α3.7 (24.37%) and αpolyA2(A>G) (18.12%). Hematological parameters such as Hb, MCV, MCH and HbH were assessed and results showed that they varied significantly among genotypes, adjusted to age and gender. This study reveals a highly diverse range of HbH patients different from what was thought in terms of both genotype and phenotype in the Khuzestan region of Iran. These findings could contribute to improve the thalassemia managing policies in this province.

Development of a novel multiplex polymerase chain reaction system for forensic individual identification using insertion/deletion polymorphisms.

Insertion/deletion (InDel) polymorphisms have been widely used in the fields of population genetics, genetic map constructions, and forensic investigations owing to the advantages of their low mutation rates, widespread distributions in the human genome, and small amplicon sizes. In order to provide more InDels with high discrimination power in Chinese populations, we selected and constructed one novel multiplex PCR-InDel panel for forensic individual identification. Genetic distributions of these 35 InDels in five reference populations from East Asia showed low genetic differentiations among these populations. Forensic efficiency evaluations of these InDels revealed that these loci could perform well for forensic individual identifications in these reference populations. In the meantime, genetic diversities and forensic parameters of these InDels were further investigated in the studied Kazak group. Mean value of polymorphism information content for 35 InDels was 0.3611. Cumulative power of discrimination of 35 InDels was 0.99999999999999603 in Kazak group. Given these results, the panel is suitable for individual identifications in the studied Kazak and these reference populations.

Multiplex PCR system for fungal pathogen detection

Introduction: The rapid and correct identification of pathogenic species is crucial for several reasons that differ according to the area of interest. In clinical laboratories it is important since knowing the pathogen guides appropriate treatment, dose and duration of therapy. In clinical microbiology laboratories culture and microscopic examination remain the “gold standard”. Biochemical and mass spectometry identification systems, such as the Vitek systems (BioMerieux®) or MALDI-TOF are examples of commercial systems for microbial identification with the advantage of being semi automatic. However, these methods are based on the regrowth and isolation of the microorganism from samples, which in some cases, such as from blood, is time consuming and the success rate of cultivation can be as low as 20%. Recently, several techniques based on the polymerase chain reaction (PCR) have been developed for microbial pathogenic detection and identification particularly Real-Time PCR methods. In this study we developed an alternative PCR based method for the detection of fungal pathogens involved in systemic infections. This is a multiplex PCR method that attributes to each species specific amplicon lengths and a fluoresce dye, according to previously design panels, enabling a fast and reproducible co-amplifying of several loci in a single PCR reaction. Materials and methods: First a panel for species identification, combining fluorescence with molecular weight of specific PCR fragments for Candida and Asprgillus was designed. The PCR fragments obtained are then analysed by capillary electrophoresis and GeneScan fragment analysis. This methodology was optimized using DNA extracted from strains previously identified and, in order to optimize the methodology to clinical samples, we also used serum from healthy donors spiked with different concentrations of fungal DNA. Then the method was tested in DNA extracted from different types of clinical samples, including blood, biopsies and bronchoalveolar lavages of patients with invasive fungal infections (IFI). Results: The optimization of the method, by using DNA from known strains belonging to the target species and strains form other species, showed 100% of specificity. The calculated yield from DNA extracted from serum spiked with fungal DNA was of around 80%. This DNA was then used to determine the sensitivity of the technique and results showed that we were able to obtain amplification products within a range of 1 to 10 pg of the total DNA extracted. Results obtained with DNA extracted from samples of IFI patients, showed that we were able to detect the specific fungal species in 75% of the samples. Several optimizations are being performed. Discussion and conclusions: This new methodology is a promising method since interpretation of results is easy, based on presence/absence of a particular peak of the panel, it is fast, accurate and reproducible and due to the design of the identification panel it is able to identify the pathogenic fungal species involved in mixed infections. Several optimizations are being performed to enhance sensitivity of the method.

SNP Typing Using Multiplex Real-Time PCR Assay for Species Identification of Forensically Important Blowflies and Fleshflies Collected in South Korea (Diptera: Calliphoridae and Sarcophagidae).

Medicolegal entomology—a subfield of forensic entomology—is mainly used in medicolegal investigations to estimate the postmortem interval (PMI). The minimum PMI of a corpse invaded by necrophagous immature insects can be estimated because the PMI is near to or earlier than the oviposition time of the larvae that hatched and fed on the corpse. As the growth speeds of larvae differ depending on temperature and species, species-specific growth data are used to estimate the minimum PMI. While morphological identification of adult necrophagous flies can be done by a well-trained entomologist, identification of larvae is relatively difficult. Larvae can only be identified up to the family level and developmental stage by observing the posterior spiracles. For these reasons, the molecular biology method of DNA barcoding has been developed. DNA barcoding that targets the mitochondrial cytochrome c oxidase subunit I (COI) gene is commonly used. COI sequences are currently acquired using polymerase chain reaction (PCR) and Sanger sequencing, which are too time-consuming and complex for practical use in medicolegal investigations. To compensate for these limitations and facilitate the use of entomology for medicolegal investigation, we designed a multiplex real-time PCR system to identify nineteen forensically important species of Calliphoridae and Sarcophagidae flies collected in South Korea. In contrast to the Sanger nucleotide sequencing process, this technology only requires a one-step real-time PCR with melt curve analysis of amplicons generated by primers targeting species-specific single nucleotide polymorphisms (SNPs). Multiplex real-time PCR was performed for twelve species of Calliphoridae (four reactions) and for seven species of Sarcophagidae (three reactions). This assay is expected to make it easier and faster for investigating authorities to identify major species of necrophagous flies at beginning of investigation and to increase the utilization of entomological evidence in forensic investigations.

Comparison of Two Multiplex PCR Systems for Meat Species Authentication

Background: Meat species adulteration has become a problem of concern. This study aimed to compare two previously published multiplex Polymerase Chain Reaction (PCR) methods for meat species authentication. Methods: The primers used in the first multiplex PCR involved species-specific reverse primer for sheep, goat, cattle, pig, and donkey with universal forward primer. In the second multiplex PCR, the primers included species-specific forward and reverse primer for pork, lamb, ostrich, horse, and cow. The extracted DNA was then amplified with species-specific primers and with mix primers separately in the respective multiplex PCR. Results: The first multiplex PCR was accompanied with cross reactivity, whereas the second multiplex PCR was specific as expected for pork, lamb, ostrich, horse, and cow. The first set of multiplex PCR showed not always amplification of all species-specific DNAs with a mixture of DNA from mentioned animals. Regarding the second set of primers, the extracted DNA of different meat species was amplified with corresponding species primers as simplex PCR resulting in specific amplicons for species DNA prepared from sheep, ostrich, horse, pig, and cattle with the specific PCR products of 119, 155, 253, 100, and 311 bp, respectively. Conclusion: Based on the present investigation, we recommend the multiplex PCR with the second set of primers included species-specific forward and reverse primers for species authentication of five meat types, including pork, lamb, ostrich, horse, as well as cow.

New fusion sarcomas: histopathology and clinical significance of selected entities

Many sarcomas contain gene fusions that can be pathogenetic mechanisms and diagnostic markers. In this article we review selected fusion sarcomas and techniques for their detection. CIC-DUX4 fusion sarcoma is a round cell tumor now considered an entity separate from Ewing sarcoma with a more aggressive clinical course, occurrence in older age, and predilection to soft tissues. It is composed of larger cells than Ewing sarcoma and often has prominent necrosis. Nuclear DUX4 expression is a promising immuno histochemical marker. BCOR-CCNB3 fusion sarcoma is cyclin B3-positive, usually occurs in bone or soft tissue of children, and may mimic a poorly differentiated synovial sarcoma. EWSR1-NFATC2 sarcoma may present in bone or soft tissue. It is typically composed of small round cells in a trabecular pattern in a myxoid matrix resembling myoepithelioma. ACTB-GLI1 fusion sarcoma may mimic a skin adnexal carcinoma, showing focal expression of epithelial markers and S100 protein. NTRK-fusion sarcomas include, in addition to infantile fibrosarcoma with ETV6-NTRK3 fusion, LMNA-NTRK1 fusion sarcoma, a low-grade spindle cell sarcoma seen in peripheral soft tissues in children and young adults. Methods to detect gene fusions include next-generation sequencing panels, anchored multiplex polymerase chain reaction systems to detect partner for a known fusion gene, and comprehensive RNA sequencing to detect virtually all gene fusions. In situ hybridization testing using probes for both fusion partners can be used as an alternative confirmation technique, especially in the absence of satisfactory RNA yield. In addition, fusion protein-related and other immunohistochemical markers can have a high specificity for fusion sarcomas.

A multiplex reverse transcription PCR assay for simultaneous detection of six main RNA viruses in tomato plants

Tomato virus diseases occur all around the world, causing serious yield losses. To detect these viruses quickly and provide a basis for disease control, a multiplex reverse transcription polymerase chain reaction system was established for simultaneous detection of Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato chlorosis virus (ToCV), Potato virus Y (PVY) and Potato virus X (PVX) in tomato plants, with 6 pairs of specific primers being designed based on the coat protein (CP) genes of these viruses. Transcriptional elongation factor-1α (EF-1α) from tomato was added to the multiplex RT-PCR reaction system to prevent false negatives. The concentration of the primers, annealing temperature, annealing time, extension time and amplification cycles were optimized. Expected fragments of 159 bp (ToCV), 262 bp (PVY), 362 bp (EF-1α), 430 bp (TMV), 500 bp (TSWV), 600 bp (CMV) and 705 bp (PVX) were amplified by this multiplex RT-PCR system, and their origin was confirmed by DNA sequencing. This method will have a wide application in virus detection of field samples.