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Abstract Introduction. El Alférez, a village in Los Montes de María (Bolívar, Colombia) and a macro-focus of leishmaniasis, recorded its first case in 2018, evidencing changes in the distribution and eco-epidemiology of the disease, although interactions between vectors and local fauna remain unknown. Objective. To evaluate the diversity of sandflies and their blood meal sources in the community of El Alférez in the municipality of El Carmen de Bolívar (Bolívar, Colombia). Materials and methods. In 2018, sandflies were collected using LED-based light traps in domestic, peridomestic, and sylvatic ecotopes and identified at the species level. Multiplex polymerase chain reaction targeting the mitochondrial cytochrome B gene was used to analyze blood from the digestive tract. Results. Lutzomyia evansi was the most abundant species (71.85%; n = 485/675), followed by Lu. panamensis, Lu. gomezi, Lu. trinidadensis, Lu. dubitans, Lu. abonnenci, and Lu.aclydifera. Twenty-five percent of the species had blood meals from Canis familiaris (36.00%; n = 9/25), Ovis aries (36.00%; n=9:/25), Bos taurus (24.00%; n = 6/25), Sus scrofa (20.00%; n = 5/25), and Homo sapiens (8.00%; n = 2/25). Lutzomyia evansi registered the highest feeding frequency (68.00%; n = 17/25), predominantly on a single (44.00%; n = 11/25) or multiple species (24.00%; n = 6/25). Conclusion. Results indicate a eclectic feeding behavior in Lu. evansi, implying potential reservoir hosts for Leishmania spp. and increasing transmission risk. This study is a first step towards understanding the diversity of mammalian blood sources used by sandflies, that may be crucial for vector identification and formulation of effective control measures.
Resumen Introducción. En 2018, en la vereda El Alférez de Los Montes de María (Bolívar, Colombia), un macrofoco de leishmaniasis, se reportó el primer caso y se evidenciaron cambios en la distribución y ecoepidemiología de la enfermedad. No obstante, las interacciones entre vectores y fauna local aún son desconocidas. Objetivo. Evaluar la diversidad de flebotomíneos y sus fuentes de alimentación sanguínea en la comunidad de El Alférez del municipio de El Carmen de Bolívar (Bolívar, Colombia). Materiales y métodos. En el 2018, se recolectaron flebotomíneos mediante trampas de luz led ubicadas en el domicilio, el peridomicilio y en el área silvestre, y se identificaron a nivel de especie. Se utilizó la reacción en cadena de la polimerasa múltiple dirigida al gen mitocondrial citocromo B para analizar la sangre del aparato digestivo. Resultados. Lutzomyia evansi fue la especie más abundante (71,85 %; n = 485/675), seguida por Lu. panamensis, Lu. gomezi, Lu. trinidadensis, Lu. dubitans, Lu. abonnenci y Lu. aclydifera. El 25 % (n = 25/100) de las especies analizadas tuvieron como fuentes de ingesta sanguínea a Canis familiaris (36 %; n = 9/25), Ovis aries (36 %; n = 9/25), Bos taurus (24 %; n = 6/25), Sus scrofa (20 %; n = 5/25) y Homo sapiens (8 %; n = 2/25). Lutzomyia evansi fue la especie con la mayor frecuencia de alimentación (68 %; n = 17/25), predominantemente de una sola especie (44 %; n = 11/25) o de varias (24 %; n = 6/25). Conclusiones. Los hallazgos indican un comportamiento alimenticio ecléctico en Lu. evansi que implica potenciales reservorios para Leishmania spp. y eleva el riesgo de transmisión. Este estudio es un primer paso para comprender la diversidad de fuentes sanguíneas de mamíferos, utilizadas por los flebotomíneos, y que pueden ser cruciales para identificación de vectores y la formulación de medidas de control eficaces.
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@#Objective To develop and verify a multiplex fluorescent quantitative PCR method for detection of common bacterial and fungal contaminants in cell culture medium.Methods According to NUC gene of Staphylococcus aureus,COLA gene of Clostridium spore,ITS-2 segment sequence of Candida albicans,a set of primers and probes were designed for each respectively,and using UBI3 gene of capsicum introduced as external standard gene,a triple reaction system of Staphylococcus aureus,Clostridium spore and external standard gene and a double reaction system of Candida albicans and external standard gene were established.The primer specificity,linear range,limit of detection,specificity,anti-interference performance and precision of the method were verified.Finally,100 samples of 293T cell culture medium were detected by using the developed method,which was compared with the common PCR method.Results Three pairs of primers all amplified about 100 bp specific gene bands corresponding to the three strains at different annealing temperatures(56,57,58 and59 ℃),and the size was consistent with the expected.In the range of 5.80 × 10~6 — 5.80 × 10~2 copies/μL,the standard plasmids of the three strains showed a good linear relationship with the Ct values.The standard curve equations were:Y=-3.373 X+37.48,Y=-3.557X+36.59 and Y=-3.536 X+39.78,each R~2> 0.99,respectively,and the amplification efficiency was in the range of 90%—110%.All the limits of detection of the three strains were 10~1 CFU/mL.The primers and probes of the three strains showed no specific amplification on the genomic DNA of six kinds of cells that were prone to cross-reaction.The genomic DNA of 293T cells,Yeast,Escherichia coli and Mycoplasma sp.had no effect on the detection.The CVs of repeatability and intermediate precision verification were both less than 15%.Among 100 cell culture medium samples,14 positive and 86 negative samples were detected,and the results of common PCR method for three positive and two negative samples randomly selected were consistent with the developed method.Conclusion The multiplex fluorescent quantitative PCR method developed in this study for the detection of bacteria and fungi in cell culture medium has good specificity,anti-interference performance and precision,and is simple to operate with low cost and high sensitivity,which can quickly detect the contaminants during cell culture.
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To address the throughput limitations of digital nucleic acid analysis,a tricolor combination-based droplet coding technique was developed to achieve multiplex digital nucleic acid analysis with flexible throughput expansibility.To improve the analysis efficiency,a machine learning-based method was further developed for automatic decoding of color-coded droplet array.The machine learning algorithm empowered the computer program to automatically extract the color-position-quantity information of the droplets.By correlating this color-position-quantity of droplets before and after nucleic acid amplification,the proportion of positive droplets for each target was rapidly determined.This droplet decoding strategy was applied to multiplex digital nucleic acid analysis.The experimental results demonstrated that this droplet decoding method was fast and accurate,with a decoding process completed within 2 min.Furthermore,the droplet identification accuracy exceeded 99%.Additionally,the obtained nucleic acid quantification results exhibited a good correlation(R2＞0.99)with those reported by a commercial digital PCR instrument.
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Objective To investigate the association of 13 single nucleotide polymorphism(SNP)sites in 6 phalange-bone development related genes[fibroblast growth factor receptor 2(FGFR2),indian hedgehog signaling molecule(IHH),Msh homeobox 1(MSX1),Runx family transcription factor 2(RUNX2),SRY-box transcription factor 9(SOX9),Wnt family member 5A(WNT5A)]with human index-ring finger length ratio(2D∶4D).Methods Digital cameras were used to take frontal photographs of the hands of 731 college students(358 males and 373 females)in Ningxia,and image analysis software was used to mark anatomical points and measure finger lengths of index(2th)and ring(4th);genotyping of 13 SNP sites(rs1047057,rs755793,rs41258305,rs3731881,rs3100776,rs12532,rs3821949,rs45585135,rs3749863,rs1042667,rs12601701,rs1829556,rs3732750)for 6 genes by multiplex PCR;One-Way ANOVA or independent sample t-test indirectly assessed the association between 2D∶4D and 13 SNP sites.Results Both left and right hand 2D∶4D were significantly higher in females than males in Ningxia college students(all P<0.01);no statistically significant differences in genotype and allele frequencies of the 13 SNP sites among different sexes(all P>0.05);among different sexes,male left hand 2D∶4D was significantly associated with the genotype of SOX9 gene rs12601701 site(P<0.05)and right hand 2D∶4D was significantly associated with the genotype of WNT5A gene rs1829556 site(P<0.05);the female right hand 2D∶4D was significantly associated with the MSX1 gene rs12532(P<0.01)and rs3821949(P<0.05)sites genotypes.Conclusion SOX9(rs12601701),WNT5A(rs1829556)and MSX1(rs12532 and rs3821949)gene polymorphisms may be associated with the formation of 2D∶4D in Ningxia population.
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Aims@#This study aimed to detect bacterial pathogens that cause sexually transmitted diseases (STD) using multiplex polymerase chain reaction and reverse hybridization.@*Methodology and results@#Thirty urine samples were collected from male patients aged between 20 and 45 in Dohuk City who were suspected of having an STD. The samples were tested for the presence of five main types of bacteria, namely Ureaplasma urealyticum, Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium and Chlamydia trachomatis responsible for causing STDs. Nineteen of the thirty urine samples were positive for at least one of the five species of bacteria, yielding a positive rate of 63.3%. Ureaplasma urealyticum had the highest diagnostic rate of 68.4% among positive samples, while C. trachomatis had the lowest diagnosis rate of 5.2%. Both N. gonorrhoeae and M. hominis had a 15.7% diagnosis rate, while M. genitalium had a 10.5% diagnosis rate. @*Conclusion, significance and impact of study @#Research findings suggest that U. urealyticum was the most common cause of STD, accounting for 68.4% of the positive samples. Conversely, the study identifies C. trachomatis as the least prevalent cause, accounting for only 5.2% of the cases. These noteworthy findings shed light on the prevalence of these bacterial pathogens in sexually transmitted diseases, laying the groundwork for more precise and effective diagnostic and treatment options.
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ObjectiveInferring cancer driver genes, especially rare or sample-specific cancer driver genes, is crucial for precision oncology. Considering the high inter-tumor heterogeneity, a few recent methods attempt to reveal cancer driver genes at the individual level. However, most of these methods generally integrate multi-omics data into a single biomolecular network (e.g., gene regulatory network or protein-protein interaction network) to identify cancer driver genes, which results in missing important interactions highlighted in different networks. Thus, the development of a multiplex network method is imperative in order to integrate the interactions of different biomolecular networks and facilitate the identification of cancer driver genes. MethodsA multiplex network control method called Personalized cancer Driver Genes with Multiplex biomolecular Networks (PDGMN) was proposed. Firstly, the sample-specific multiplex network, which contains protein-protein interaction layer and gene-gene association layer, was constructed based on gene expression data. Subsequently, somatic mutation data was integrated to weight the nodes in the sample-specific multiplex network. Finally, a weighted minimum vertex cover set identification algorithm was designed to find the optimal set of driver nodes, facilitating the identification of personalized cancer driver genes. ResultsThe results derived from three TCGA cancer datasets indicate that PDGMN outperforms other existing methods in identifying personalized cancer driver genes, and it can effectively identify the rare driver genes in individual patients. Particularly, the experimental results indicate that PDGMN can capture the unique characteristics of different biomolecular networks to improve cancer driver gene identification. ConclusionPDGMN can effectively identify personalized cancer driver genes and broaden our understanding of cancer driver gene identification from a multiplex network perspective. The source code and datasets used in this work are available at https://github.com/NWPU-903PR/PDGMN.
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@#Objective To develop and verify a multiplex fluorescent quantitative PCR method for detection of common bacterial and fungal contaminants in cell culture medium.Methods According to NUC gene of Staphylococcus aureus,COLA gene of Clostridium spore,ITS-2 segment sequence of Candida albicans,a set of primers and probes were designed for each respectively,and using UBI3 gene of capsicum introduced as external standard gene,a triple reaction system of Staphylococcus aureus,Clostridium spore and external standard gene and a double reaction system of Candida albicans and external standard gene were established.The primer specificity,linear range,limit of detection,specificity,anti-interference performance and precision of the method were verified.Finally,100 samples of 293T cell culture medium were detected by using the developed method,which was compared with the common PCR method.Results Three pairs of primers all amplified about 100 bp specific gene bands corresponding to the three strains at different annealing temperatures(56,57,58 and59 ℃),and the size was consistent with the expected.In the range of 5.80 × 10~6 — 5.80 × 10~2 copies/μL,the standard plasmids of the three strains showed a good linear relationship with the Ct values.The standard curve equations were:Y=-3.373 X+37.48,Y=-3.557X+36.59 and Y=-3.536 X+39.78,each R~2> 0.99,respectively,and the amplification efficiency was in the range of 90%—110%.All the limits of detection of the three strains were 10~1 CFU/mL.The primers and probes of the three strains showed no specific amplification on the genomic DNA of six kinds of cells that were prone to cross-reaction.The genomic DNA of 293T cells,Yeast,Escherichia coli and Mycoplasma sp.had no effect on the detection.The CVs of repeatability and intermediate precision verification were both less than 15%.Among 100 cell culture medium samples,14 positive and 86 negative samples were detected,and the results of common PCR method for three positive and two negative samples randomly selected were consistent with the developed method.Conclusion The multiplex fluorescent quantitative PCR method developed in this study for the detection of bacteria and fungi in cell culture medium has good specificity,anti-interference performance and precision,and is simple to operate with low cost and high sensitivity,which can quickly detect the contaminants during cell culture.
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Objective To investigate the association between the index finger and ring finger length ratio (2D ∶ 4D) and of four loci (rs6461992‚ rs6968828‚ rs7801581‚ rs17427875) polymorphism of homeobox (HOX) A11 gene among Ningxia college students. Methods Digit camera was used to collect frontal hand photos of 667 Han college students (348 males and 319 females) from Ningxia province; Image analysis software was used to mark the anatomical points and measure finger lengths of the index and ring fingers of both hands; multiplex PCR was used to detect each locus polymorphisms of HOXA11 gene; statistical software was used to compare and analyze the differences and associations of 2D ∶4D and gene polymorphisms between different genders. Results Among Ningxia Han college students‚ both left hand and right hand 2D ∶ 4D were significantly higher in females than those of in males (all P< 0. 05)‚ and there were no significant sex differences in right-left hand 2D ∶4D; the genotypes and allele frequencies of rs7801581 locus of HOXA11 gene differed significantly between genders (all P < 0. 05)‚ and none of the other locus polymorphisms showed any significant sex differences; only female left hand 2D ∶4D was significantly associated with rs6461992 locus genotype in the relationship between 2D ∶4D and HOXA11 polymorphisms (P<0. 05). Conclusion There were significant sex differences in 2D ∶ 4D among Han college students in Ningxia‚ and the rs6461992 locus polymorphism of HOXA11 gene may be associated with the formation of 2D ∶4D in females.
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Resumen La amplificación de sondas múltiples dependientes de ligación (MLPA) es una valiosa herramienta en el estudio de alteraciones en el número de copias para distintas patologías de origen genético. La existencia de una amplia oferta de kits comerciales, el fácil y rápido procesamiento de laboratorio, su alta sensibilidad y, en general, los buenos resultados informados han permitido que su uso se encuentre expandido en muchos laboratorios de biología molecular alrededor del mundo. El principio de esta técnica ha sido adaptado para distintas aplicaciones como la MLPA metilación específica para el estudio de enfermedades relacionadas con la impronta epigenética y la MLPA digital que se acopla a equipos de secuenciación de nueva generación para aumentar la cantidad de regiones analizadas en un solo ensayo. Al contar con 10 años de experiencia en el uso de esta técnica en el Laboratorio Nacional de Tamizaje Neonatal y Alto Riesgo se realiza esta revisión con el fin de analizar los principios, variantes de la técnica, análisis de los datos, algunas aplicaciones, ventajas y desventajas de la MLPA en comparación con otras tecnologías disponibles.
Abstract Multiplex ligation-dependent probe amplification (MLPA) is a valuable tool in the study of copy number alterations for different pathologies of genetic origin. The availability of a wide range of commercial kits, the easy and fast laboratory processing, its high sensitivity and, in general, the good results reported have allowed its use to be expanded in many molecular biology laboratories around the world. The basic principle of this technique has been adapted for different applications such as specific methylation MLPA for the study of diseases related to epigenetic imprinting and digital MLPA that is coupled to next-generation sequencing equipment to increase the number of regions analysed in a single trial. With 10 years of experience in the use of this technique, the National Laboratory for Neonatal and High Risk Screening performs this review in order to analyse the principles, variants of the technique, data analysis, some applications, and advantages and disadvantages of MLPA compared to other available technologies.
Resumo A amplificação de múltiplas sondas dependentes de ligação (MLPA) é uma ferramenta valiosa no estudo das alterações do número de cópias para diferentes patologias de origem genética. A existência de uma ampla gama de kits comerciais, processamento laboratorial fácil e rápido, alta sensibilidade e, em geral, os bons resultados relatados, permitiram que seu uso se encontre expandido em muitos laboratórios de biologia molecular ao redor do mundo. O princípio desta técnica foi adaptado para diferentes aplicações como a MLPA metilação específica para o estudo de doenças relacionadas com o imprinting epigenético e a MLPA digital que é acoplada a equipamentos de sequenciamento de nova geração para aumentar o número de regiões analisadas em um único ensaio. Com 10 anos de experiência no uso da técnica, o Laboratório Nacional de Triagem Neonatal e de Alto Risco realiza esta revisão visando a analisar os princípios, variantes da técnica, análise dos dados, algumas aplicações, vantagens e desvantagens da MLPA comparado com outras tecnologias disponíveis.
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Resumen Antecedentes: Las infecciones de transmisión sexual son un problema de salud pública mundial. El análisis rutinario incluye solo pruebas microbiológicas y serológicas para el diagnóstico de patógenos. Los microorganismos atípicos como Chlamydia trachomatis y micoplasmas no son identificados debido a los requerimientos. Además, no es incluida Gardnerella vaginalis, aunque se asocia a la vaginosis bacteriana. Objetivo: Desarrollar una PCR múltiplex para el diagnóstico de C. trachomatis, micoplasmas y G. vaginalis. Método: Se estandarizó la PCR múltiplex utilizando oligonucleótidos para C. trachomatis (gen ompA, orf6 plasmídico), Mycoplasma/Ureaplasma y G. vaginalis (genes rRNA16s). Resultados: Se estandarizaron pruebas de PCR múltiplex para los microorganismos estudiados, optimizándose las concentraciones y condiciones de las reacciones múltiplex. Se obtuvieron PCR dúplex para C. trachomatis (ompA, orf6), Chlamydia/Gardnerella y Chlamydia/micoplasmas y tríplex para Chlamydia/Mycoplasma/Ureaplasma. También un cuádruplex para Chlamydia/Mycoplasma/Ureaplasma/Gardnerella. Los resultados fueron verificados por PCR e hibridación automática (HybriSpot 12) y análisis in silico. Conclusión: Se desarrollaron pruebas de PCR múltiplex con una alta sensibilidad y especificidad para la identificación de C. trachomatis, micoplasmas y G. vaginalis.
Abstract Background: Sexually transmitted infections are a global public health problem. Routine analysis includes microbiological and serological tests for the diagnosis of pathogens. Atypical microorganisms such as Chlamydia trachomatis and mycoplasmas are not determined due to the requirements for their identification. Furthermore, Gardnerella vaginalis is not included despite being associated with bacterial vaginosis. Objective: To develop a multiplex PCR to diagnose Chlamydia, mycoplasmas, and Gardnerella. Method: Standardization of multiplex PCR tests was carried out using oligonucleotides for the identification of Chlamydia (ompA gene, plasmid orf6), Mycoplasma/Ureaplasma and Gardnerella (rRNA16s genes). Results: Multiplex PCR tests were standardized for the microorganisms studied, optimizing the concentrations and conditions of the multiplex reactions. Duplex PCR was obtained for Chlamydia (ompA, orf6), Chlamydia/Gardnerella, and Chlamydia/mycoplasmas, and triplex PCR for Chlamydia/mycoplasmas. Also, a quadruplex for Chlamydia, Mycoplasma/Ureaplasma and Gardnerella. PCR and automatic hybridization verified the results obtained (HybriSpot 12) and in silico analysis. Conclusion: Multiplex PCR tests with high sensitivity and specificity were developed to identify C. trachomatis, mycoplasmas, and G. vaginalis.
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INTRODUCCIÓN: La diarrea aguda continúa siendo una de las principales causas de morbilidad en niños; sin embargo, el diagnóstico etiológico presenta limitaciones dada la baja sensibilidad de los métodos tradicionales. OBJETIVO: Describir los microorganismos identificados en niños que acudieron al Servicio de Urgencia (SU) de un hospital universitario en Santiago, Chile, por diarrea aguda y a los que se le solicitó panel molecular gastrointestinal. MÉTODOS: Se revisaron fichas clínicas y resultados de panel gastrointestinal realizados entre junio de 2017 y marzo de 2020. RESULTADOS: Se incluyeron 198 pacientes, edad promedio de 54,5 meses y 60,6% (120/198) de sexo masculino. La positividad del panel fue de 78,8% (156/198) con 35,3% (55/156) de las muestras polimicrobianas. Se identificaron 229 microorganismos, de los cuales 72,9% (167/229) corresponden a bacterias, 25,8% (59/229) a virus y 1,3% (3/229) a parásitos. Destacaron Campylobacter spp. y Escherichia coli enteropatógena (ECEP) como las bacterias más frecuentemente identificadas. Los pacientes con detección de Campylobacter spp. presentaron con mayor frecuencia fiebre (p = 0,00). ECEP se aisló principalmente (82,5%) en muestras polimicrobianas. DISCUSIÓN: Los resultados enfatizan el potencial que poseen los estudios moleculares para mejorar el diagnóstico etiológico de la diarrea, pero a la vez llevan a cuestionar el rol patogénico de algunos microorganismos identificados.
BACKGROUND: Acute diarrhea continues to be one of the main causes of morbidity in children, however the etiologica diagnosis presents limitations given the low sensitivity of traditional methods. AIM: To describe the microorganisms identified in children who attended the emergency department (ED) in Santiago, Chile, due to acute diarrhea and to whom a gastrointestinal panel was requested as part of their study. MATERIAL AND METHODS: Clinical records and results of the gastrointestinal panel carried out between June 2017 and March 2020 were reviewed. RESULTS: 198 patients were included, the average age was 54.5 months and 60.6% (120/198) were males. Positivity was 78.8% (156/198) with 35.3% (55/156) of the samples being polymicrobial. 229 microorganisms were identified, of which 72.9% (167/229) corresponded to bacteria, 25.8% (59/229) to viruses, and 1.3% (3/229) to parasites. Campylobacter spp. and enteropathogenic Escherichia coli (EPEC) were the most frequently identified bacteria. Patients with detection of Campylobacter spp. presented a higher frequency of fever (p = 0.00). EPEC was isolated in 82.5% of the cases in polymicrobial samples. DISCUSSION: The results emphasize the potential of molecular studies to improve the etiological diagnosis of diarrhea and at the same time lead to question the pathogenic role of some microorganisms.
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Humanos , Masculino , Femenino , Diarrea/diagnóstico , Heces/microbiología , Parásitos/aislamiento & purificación , Estaciones del Año , Bacterias/aislamiento & purificación , Virus/aislamiento & purificación , Chile , Estudios Retrospectivos , Diarrea/etiología , Diarrea/epidemiología , Servicio de Urgencia en Hospital , Heces/parasitologíaRESUMEN
Background: The quinolone group, a synthetic antimicrobial, is widely used worldwide to treat many diseases, including those caused by Gram-negative bacteria. Escherichia coli and others are among the bacteria that produce quinolone resistance genes (qnr) such as qnrA and aac(6?)-Ib-cr. Objective: The present study aimed to the isolate Escherichia coli from patients attending some Hospitals in Wad Medani city, identification of drug resistance patterns and detection of the frequency of quinolones resistance genes; qnrA and aac(6?)-Ib-cr among isolated strains. Methods: A cross-sectional descriptive, hospital-based study included 119 Escherichia coli strains was conducted. A designed questionnaire used for demographic data collection and the attitude toward antimicrobials usage. Clinical specimens were processed for aerobic bacterial isolation and identification. Antimicrobial sensitivity performed by Kirby Bauer disc diffusion technique according to the CLSI guidelines. Presence of qnrA and aac(6?)-Ib-cr genes was assessed by multiplex PCR. Results: Most strains of Escherichia coli originated from urine 53.8% (64/119) and wounds 42.9% (51/119) specimens. Meropenem had the best effect against tested strains with susceptibility of 85% (101/119). Multiplex PCR assay, using specific primers, demonstrated that 41.2% (49/119) and 37.8% (45/119) of isolated Escherichia coli possessed qnrA and aac(6?)-Ib-cr genes respectively. Conclusion: The high rate of qnrA and aac (6)-Ib-cr genes among Escherichia coli necessitate the usage of molecular tools in detecting the genetic determinants of drug resistance microorganisms in countries such as Sudan.
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Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA)signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 µL PCR reaction includes primer(s) (0.3–0.5 µM each), dNTP mixture (160 µM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 µL), DNA template (250 ng/50 µL), BSA (0.4 µg/µL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics.
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@#Mass-encoded probe is a probing tool that specifically identifies target molecules and thus outputs their characteristic ion signals with mass tags.It plays an important role in multiplex assay of disease markers, drug target screening and other biomedical applications.Based on various mass spectrometric methods, researchers have developed an array of mass tag-encoded probes with different structures and functions, providing powerful technical tools for multiplex detection of biomolecules in physiological environments and for mass spectrometry imaging of tissue samples.This review introduces the latest research progress of mass tag-encoded probes in multiplex mass spectrometric detection from three aspects, i.e. structural composition of the probes, mass spectrometric methods and their application in biochemical analysis, with a prospect of the future development of mass tag-encoded probes.
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ObjectiveTo establish a set of single nucleotide polymorphisms (SNP) detection protocol for inbred rats based on multiplex PCR-ligase detection reaction (LDR). MethodsA total of 40 rats SNP sites were selected on chromosomes 1-20 and X of rats among 5 inbred strains of rats, and the 40 SNP sites were randomly divided into four groups. A genetic detection protocol for 4 groups of SNP in inbred rats based on multiplex PCR-LDR technology was constructed. 9 commonly used rat strains from two other domestic rat suppliers were detected by this protocol. Finally, the feasibility of this protocol was verified by comparing the amplification effects of different DNA polymerases by a third-party laboratory. ResultsWhen using the constructed SNP detection protocol for inbred rats to test 5 rat strains, all sites in each sample obtained good amplification results. The 9 commonly used rat strains from two other rat suppliers in china were also well amplified by this SNP detection protocol, and 40 SNPs were homozygous in each Inbred strain. The results of detection of the same rat DNA samples with three different DNA polymerases showed that the Multiplex PCR Kit, AmpliTaq Gold 360 DNA polymerase and Platinum II Taq hot start DNA polymerase had electrophoretic peaks of amplification products at all SNP sites in groups 1 to 3, and Platinum II Taq hot start DNA polymerase had one less electrophoretic peak of the amplification products at the SNP sites in group 4. In addition, inter-laboratory comparisons showed consistent results for the same amplification system. ConclusionBased on multiplex PCR-LDR technology, this study successfully established a SNP detection protocol for rats covering all autosomes and X chromosomes with the excellent stability and repeatability.
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@#ObjectiveTo develop and verify a multiplex real-time RT-PCR assay for simultaneous identification of human parainfluenza virus type 1(HPIV1),type 2(HPIV2)and type 3(HPIV3).MethodsThe whole genome sequences of HPIV1,HPIV2 and HPIV3 were downloaded from the database for alignment analysis,and the conserved regions were selected. Specific primers and probes were designed for the three viruses respectively to develop a multiplex real-time RTPCR assay with human ribonuclease P(RNase P)as theinternal quality control. The method was verified for the sensitivity,specificity and precision and used to detect 192 clinical samples.ResultsAfter optimization,the multiplex real-time RTPCR reaction system was determined to be 30 μL,including 10 × NeoscriptRTase/UNG Multi mix 3 μL,5 × Neoscript RT Premix Multi Buffer 6 μL,upstream and downstream primers of HPIV1,HPIV2 and HPIV3(100 μmol/L)0. 1 μL respectively,HPIV1,HPIV2,HPIV3 probes(100 μmol/L)0. 05 μL respectively,RNase P upstream and downstream primers(50 μmol/L)0. 06 μL respectively,RNase P probe(50 μmol/L)0. 03 μL respectively,template 15 μL,and ddH2O supplemented to 30 μL. The reaction conditions were 50 ℃ 20 min,95 ℃ 3 min and 45 cycles of 95 ℃ 15 s and54 ℃ 30 s. Fluorescence signals were collected during annealing in each cycle. The minimum detection limits of HPIV1,HPIV2 and HPIV3 were all 500 copies/mL by the multiplex real-time RT-PCR assay;The method showed no cross-reaction with influenza A virus,influenza B virus,respiratory syncytial virus andnovel coronaviruses. The coefficients of variation(CVs)in intra-and inter-groups of the recombinant plasmid standard mixture with three different concentrations were all less than 3%. HPIV1,HPIV2 and HPIV3 were detected in 192 clinical samples,and the positive rates were7. 81%,0. 05% and 3. 1%,respectively.ConclusionThe multiplex real-time RT-PCR assay for detection of HPIV1,HPIV2 and HPIV3 developed in this study has good sensitivity,specificity and precision,which has a high clinical application prospect in the field of rapid diagnosis and identification of HPIV.
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@#Objective To develop and verify a multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity.Methods Specific reverse transcription primers,two pairs of quantitative primers and probes were designed for the CDS sequence of telomerase catalytic subunit telomerase reverse transcriptase(TERT).After optimization of the reverse transcription primers(specific reverse transcription primers and random primers) and quantitative primers(two pairs of quantitative primer probes used alone or in combination) in the reaction system,with the primer probe of internal reference gene GAPDH,multiplex fluorescence quantitative PCR was performed in a single tube.In addition,telomerase positive standard and negative standard were prepared with 293T and MRC-5 cells respectively,and the stability and precision of the method were verified.The telomerase activity in 19 normal mesenchymal cell samples and 32 breast cancer cell samples were detected by the developed method.Results The optimum reaction system was as follows:using cDNA synthesized with specific reverse transcription primers as the template,2 pairs of quantitative primer probes of TERT gene were mixed with internal reference gene GAPDH primer probes for multiplex fluorescence quantitative PCR reaction in a single tube.After optimization,the sensitivity and TERT fluorescence signal quantity of the system were greatly improved,and the ΔRn was enhanced by 3 times.The amplification curve of positive standard TERT gene was normal,and the ΔCt between TERT gene and GAPDH gene remained stable.The amplification curve of GAPDH gene in negative standard was normal,while there was no amplification curve of TERT gene.There was a little difference in ΔCt between TERT and GAPDH genes in the positive standard frozen and thawed for 3 and 5 times repeatedly and the positive standard without freezing and thawing,and the CVs of precision in intra-and inter-groups were all less than 1%.Telomerase activity was negative in 19 normal mesenchymal cell samples and positive in 32 breast cancer cell samples,and significant difference in Ct value of TERT gene between them was observed(t=4.236,P <0.001).Conclusion The developed multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity has good stability and precision,which is expected to be used in early diagnosis and gene therapy of tumors.
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Objective To investigate the association between 8 single-nucleotide polymorphisms (SNPs) of hydroxysteroid dehydrogenase (HSD) gene family and human digit ratio (2D ∶ 4D). Methods Randomly selected 808 college students (400 males and 408 females) as subjects, the digit ratio of left and right fingers were measured and calculated using computer image software. Eight SNPs (rs1000283, rs2236903, rs5479, rs56303414, rs676387, rs4445895, rs2066474, rs8190478) in HSD11B and HSD17B gene families were genotyped by multiplex PCR. The association between 2D ∶4D and different genotypes was analyzed by One-Way ANOVA. Results Female left hand(L)2D ∶ 3D, L2D ∶4D, L3D ∶4D, right hand(R)2D ∶4D, R2D ∶5D were significantly higher than male (P0. 05). The genotypes frequency of the 8 SNPs were not significantly associated with digit ratio (2D ∶4D) in both males and females (P>0. 05). Conclusion There are significant gender differences in digit ratio in Ningxia Han college students, but there is no correlation between digit ratio and 8 SNPs in HSD11B and HSD17B gene families, suggesting that HSD11B and HSD17B gene families may have nothing to do with the formation of human digit ratio.
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Objectives:To analyze the effect of vertebral or intraspinal abnormalities on the efficacy of pos-terior corrective surgery for scoliosis patients with arthrogryposis multiplex congenita(AMC).Methods:A retro-spective study was conducted on 30 scoliosis patients with AMC who underwent posterior corrective surgery in the Department of Spine Surgery of Drum Tower Hospital between August 2001 and November 2021.There were 18 males and 12 females with a mean age of 15.9±5.8(6-32)years.The patients were divided into ab-normal group(15 cases)and control group(15 cases)according to with or without vertebral or intraspinal ab-normalities.The types of vertebral or intraspinal abnormalities in the abnormal group were recorded,and the number of fusion segments,operative time and intraoperative blood loss were compared between groups.The complications during follow-up were also collected.The flexibility of major curve was assessed on Bending radiographs,and the main curve Cobb angle,the distance between C7 plumb line and center sacral vertical line(C7PL-CSVL),the sagittal vertical axis(SVA),the thoracic kyphosis(TK),and the lumbar lordosis(LL)were measured on the standing whole spine anteroposterior and lateral X-ray images at pre-operation,postoperative two weeks and two years,and the correction rate of major curve was calculated at 2 weeks after surgery and the final follow-up.Results:There were 10 cases of simple poor segmentation(66.6％),2 cases of poor seg-mentation combined with tethered cord(13.3％),and 1 case of poor segmentation combined with arachnoid cyst,simple hemivertebra,and simple wedge-shaped vertebra(6.7％)each in the abnormal group.There were no significant differences between abnormal group and control group in the number of fusion segments,opera-tive time and intraoperative blood loss(P＞0.05).No complication was observed during operation in the abnor-mal group,and 3 complications were observed during follow-up,including 2 cases with screw misplacements and 1 case with thoracic effusion and the right brachial plexus paralysis;5 cases of complications in the control group were observed,including 1 case with malignant hyperthermia and cardiac arrest during the surgery,3 cases with screw misplacements and 1 with thoracic effusion and screw placement failure.The in-cidence of complications was not statistically different between the two groups(P=0.628).The flexibility of ma-jor curve before operation was not statistically different between the two groups(P＞0.05);The major curve Cobb angle,C7PL-CSVL,SVA,TK and LL at pre-operation,post-operative two weeks and 2 months were not statistically different between groups(P＞0.05);The correction rate of major curve at 2 weeks and 2 months after surgery were not significantly different as well(P＞0.05).Conclusions:Vertebral or intraspinal abnormali-ties have no obvious effects on the efficacy of posterior corrective surgery for the treatment of scoliosis pa-tients with AMC,and there is no significant increase in the incidence of intraoperative and postoperative com-plications.
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@#The pathogenesis of chronic parasitic central nervous system (CNS) infections, including granulomatous amoebic meningoencephalitis (GAE), cerebral toxoplasmosis (CT), and neurocysticercosis (NCC), is primarily due to an inflammatory host reaction to the parasite. Inflammatory cytokines produced by invading T cells, monocytes, and CNS resident cells lead to neuroinflammation which underlie the immunopathology of these infections. Immune molecules, especially cytokines, can therefore emerge as potential biomarker(s) of CNS parasitic infections. In this study, cerebral spinal fluid (CSF) samples from suspected patients with parasitic infections were screened for pathogenic free-living amoebae by culture (n=2506) and PCR (n=275). Six proinflammatory cytokines in smear and culture-negative CSF samples from patients with GAE (n = 2), NCC (n = 7), and CT (n = 23) as well as control (n = 7) patients were measured using the Multiplex Suspension assay. None of the CSF samples tested was positive for neurotropic free-living amoebae by culture and only two samples showed Acanthamoeba 18S rRNA by PCR. Of the six cytokines measured, only IL-6 and IL-8 were significantly increased in all three infection groups compared to the control group. In addition, TNFa levels were higher in the GAE and NCC groups and IL-17 in the GAE group compared to controls. The levels of IL-1b and IFNg were very low in all the infection groups and the control group. There was a correlation between CSF cellularity and increased levels of IL-6, IL-8, and TNFa in 11 patients. Thus, quantifying inflammatory cytokine levels in CSF might help with understanding the level of neuroinflammation in patients with neurotropic parasitic diseases. Further studies with clinico-microbiological correlation in the form of reduction of cytokine levels with treatment and the correlation with neurological deficits are needed.