Academic literature on the topic 'Multiplex PCR, Co-infection, Sequencing'

This study was aimed to provide a local database for detection of coronavirus (CoV) species in suspect individual with respiratory tract infections like influenza type A and a tuberculosis using multiplex Sybr green reverse transcriptase real-time PCR (rRT-PCR) technique. A total of 500 samples was collected from individuals suffering from upper and/or lower respiratory tract diseases for testing of 4 CoV species (229E, OC43, NL63, and HKU1). RNA extracted, amplified and subsequent the positive samples sequencing. The results showed melting curve analysis (Tm) of the specific amplicons (79.73±0.36) and 9% positive for CoVs and some of them have other co-infection such as influenza virus 26.67%, and TB 11.11%. On the other hands, the CoVs were detected 4.62% in upper respiratory samples and 20.39% with lower respiratory samples. Sequencing results pointed out two isolates were CoV-NL63 and four isolates were CoV-229E, with first record accession number MN086823.1 and MN086824.1, respectively in GenBank. In conclusion, this rRT-PCR showed the rapid and efficient detection of CoVs with few copies number. This allows being used for the diagnosis of CoVs along with other respiratory viruses in a multiplex assay to reduce processing time. Subsequent applied nested RT-PCR to overcome the low viral load.

Here, a rapid and reliable two-step multiplex PCR assay for identifying 14 Gram-positive anaerobic cocci (GPAC) species originally classified in the genus Peptostreptococcus (Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus octavius, Anaerococcus prevotii, Anaerococcus tetradius, Anaerococcus vaginalis, Finegoldia magna, Micromonas micros, Peptostreptococcus anaerobius, Peptoniphilus asaccharolyticus, Peptoniphilus harei, Peptoniphilus indolicus, Peptoniphilus ivorii and Peptoniphilus lacrimalis) is reported. Fourteen type strains representing 14 GPAC species were first identified to the genus level by multiplex PCR (multiplex PCR-G). Since three of these genera (Finegoldia, Micromonas and Peptostreptococcus) contain only a single species, F. magna, M. micros and P. anaerobius, respectively, these organisms were identified to the species level directly by using the multiplex PCR-G. Then six species of the genus Anaerococcus (A. hydrogenalis, A. lactolyticus, A. octavius, A. prevotii, A. vaginalis and A. tetradius) were further identified to the species level using multiplex PCR assays (multiplex PCR-Ia and multiplex PCR-Ib). Similarly, five species of the genus Peptoniphilus (Pn. asaccharolyticus, Pn. harei, Pn. indolicus, Pn. ivorii and Pn. lacrimalis) were identified to the species level using multiplex PCR-IIa and multiplex PCR-IIb. The established two-step multiplex PCR identification scheme was applied to the identification of 190 clinical isolates of GPAC species that had been identified previously to the species level by 16S rRNA sequencing and phenotypic tests. The identification obtained from multiplex PCR assays showed 100 % agreement with 16S rDNA sequencing identification, but only 65 % (123/190) agreement with the identification obtained by phenotypic tests. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of GPAC species. It will permit a more accurate assessment of the role of various GPAC species in infection and of the degree of antimicrobial resistance in each of the group members.

Molecular diagnostics has changed the way lung cancer patients are treated worldwide. Of several different testing methods available, PCR followed by directed sequencing and amplification refractory mutation system (ARMS) are the two most commonly used diagnostic methods worldwide to detect mutations at KRAS exon 2 and EGFR kinase domain exons 18-21 in lung cancer. Compared to ARMS, the PCR followed by directed sequencing approach is relatively inexpensive but more cumbersome to perform. Moreover, with a limiting amount of genomic DNA from clinical formalin-fixed, paraffin-embedded (FFPE) specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions often get challenging. Here, we report a novel and cost-effective single multiplex-PCR based method, CRE (for Co-amplification of five KRAS and EGFR exons), followed by concatenation of the PCR product as a single linear fragment for direct sequencing. CRE is a robust protocol that can be adapted for routine use in clinical diagnostics with reduced variability, cost and turnaround time requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples. As a proof of principle, CRE is able to detect the activating EGFR L858R and T790M EGFR mutations in lung cancer cell line and primary tumors.

Molecular diagnostics has changed the way lung cancer patients are treated worldwide. Of several different testing methods available, PCR followed by directed sequencing and amplification refractory mutation system (ARMS) are the two most commonly used diagnostic methods worldwide to detect mutations at KRAS exon 2 and EGFR kinase domain exons 18-21 in lung cancer. Compared to ARMS, the PCR followed by directed sequencing approach is relatively inexpensive but more cumbersome to perform. Moreover, with a limiting amount of genomic DNA from clinical formalin-fixed, paraffin-embedded (FFPE) specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions often get challenging. Here, we report a cost-effective single multiplex-PCR based method, CRE (for Co-amplification of five KRAS and EGFR exons), followed by concatenation of the PCR product as a single linear fragment for direct sequencing. CRE is a robust protocol that can be adapted for routine use in clinical diagnostics with reduced variability, cost and turnaround time requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples. As a proof of principle, CRE is able to detect the activating EGFR L858R and T790M EGFR mutations in lung cancer cell line and primary tumors.

Abstract Background Broad testing for respiratory viruses among persons under investigation (PUI) for SARS-CoV-2 is performed inconsistently, limiting our understanding of alternative infections and co-infections in these patients. Here, we used unbiased metagenomic next-generation sequencing (mNGS) to assess the frequencies of 1) alternative viral infections in SARS-CoV-2 RT-PCR negative PUIs and 2) viral co-infections in SARS-CoV-2 RT-PCR positive PUIs. Methods A convenience sample set was selected from PUIs who were tested for SARS-CoV-2 in the Emory Healthcare system during the first 2 months of the pandemic from 02/26-04/23/20. Laboratory results were extracted by chart review; Flu/RSV and multiplex respiratory pathogen PCRs had been performed at the discretion of treating physicians. Excess nasopharyngeal swab samples were retrieved within 72 hours of collection and underwent RNA extraction and SARS-CoV-2 testing by triplex RT-PCR. mNGS was performed by DNAse treatment, random primer cDNA synthesis, Nextera XT tagmentation, and high-depth Illumina sequencing. Reads underwent taxonomic classification by KrakenUniq, as implemented in viral-ngs. Results 53 PUIs were included, 30 negative and 23 positive for SARS-CoV-2 by RT-PCR. Among SARS-CoV-2 negative PUIs, 28 (93%) underwent clinical testing for alternative infections, and 8 (29%) tested positive for another respiratory virus. In all cases, mNGS identified the same virus (Table 1). In another 3 PUIs, mNGS identified two viruses that were not tested for and one that was missed by routine testing. No SARS-CoV-2 was detected by mNGS among RT-PCR negative PUIs. Among SARS-CoV-2 RT-PCR positive PUIs, 18 (69%) underwent clinical testing for co-infections, and none were detected. mNGS did not identify any viral co-infections but did detect SARS-CoV-2 in all 23 PUIs. Table 1: Molecular and Metagenomic Testing of Persons Under Investigation Conclusion Unbiased mNGS offers the powerful opportunity to streamline testing for PUIs by assessing for SARS-CoV-2 and alternative infections simultaneously; this technique can also be used to identify co-infections, but none were observed in our study population. Interestingly, many PUIs had no infection identified on routine testing or mNGS, which may reflect inadequate sampling, rapid virus clearance, or a non-viral process. Disclosures All Authors: No reported disclosures

Context.— Infections are the leading cause of perinatal and infant mortality in low-income and low-resource countries, which have a higher prevalence of infections. Definitive diagnosis of congenital and perinatal infections is largely dependent upon the results of laboratory tests. Objective.— To develop a multiplex nested polymerase chain reaction (PCR) technique for the simultaneous detection of 7 pathogens containing DNA in their genomes in suspected cases of congenital infection. Design.— Eligible participants were pregnant women with positive immunoglobulin M antibodies raised to one of the pathogens in the prenatal serologic screening, associated or not with fetal ultrasound abnormalities or positive fetal serology. Neonates whose mothers did not attend prenatal care were included when they presented with symptomatology and laboratory parameters suggestive of infection. The detection rate of the multiplex nested PCR was compared with maternal, fetal, and neonatal serology, as well as placental immunohistochemistry and noncommercial amplifications. Results.— Of 161 suspected cases, the multiplex nested PCR detected 60 (37.3%), whereas the tests available in hospital laboratories detected 13 of 60 (21.7%) of the cases detected by the multiplex nested PCR, demonstrating a 4.6 times higher detection rate for the multiplex nested PCR (Fisher exact test, P < .001). Positive amplifications were to Toxoplasma gondii (32 cases), cytomegalovirus (14 cases), parvovirus B19 (5 cases), and adenovirus (5 cases). In 4 cases, 2 pathogens were simultaneously detected. All types of biological matrices were suitable for amplification. Sequencing of multiplex nested PCR products confirmed the molecular findings. Conclusions.— The multiplex nested PCR significantly increased the number of diagnosed congenital infections. Given the scarcity of DNA recovered from amniotic fluid and some neonatal samples, this multiplex nested PCR allows the simultaneous detection of 7 pathogens associated with congenital infections in a reliable, faster, cost-effective, and more sensitive way.

AbstractPatients with central nervous system (CNS) infection experience very high levels of morbidity and mortality, in part because of the many challenges inherent to the diagnosis of CNS infection and identification of a causative pathogen. The clinical presentation of CNS infection is nonspecific, so clinicians must often order and interpret many diagnostic tests in parallel. This can be a daunting task given the large number of potential pathogens and the availability of different testing modalities. Here, we review traditional diagnostic techniques including Gram stain and culture, serology, and polymerase chain reaction (PCR). We highlight which of these are recommended for the pathogens most commonly tested among U.S. patients with suspected CNS infection. Finally, we describe the newer broad-range diagnostic approaches, multiplex PCR and metagenomic sequencing, which are increasingly used in clinical practice.

Dogs and cats play an important role as reservoirs of vector-borne pathogens, yet reports of canine and feline vector-borne diseases in Saudi Arabia are scarce. Blood samples were collected from 188 free-roaming dogs and cats in Asir (70 dogs and 44 cats) and Riyadh (74 dogs), Saudi Arabia. The presence of Anaplasma spp., Bartonella spp., hemotropic Mycoplasma spp., Babesia spp., and Hepatozoon spp. was detected using a multiplex tandem real-time PCR. PCR-positive samples were further examined with specific conventional and real-time PCR followed by sequencing. Dogs from Riyadh tested negative for all pathogens, while 46 out of 70 dogs (65.7%) and 17 out of 44 cats (38.6%) from Asir were positive for at least one pathogen. Positive dogs were infected with Anaplasma platys (57.1%), Babesia vogeli (30%), Mycoplasma haemocanis (15.7%), and Bartonella henselae (1.4%), and cats were infected with Mycoplasma haemofelis (13.6%), Candidatus Mycoplasma haemominutum (13.6%), B. henselae (9.2%), and A. platys (2.27%), all of which are reported for the first time in Saudi Arabia. Co-infection with A. platys and B. vogeli was detected in 17 dogs (24.28%), while coinfections were not detected in cats. These results suggest that effective control and public awareness strategies for minimizing infection in animals are necessary.

Human bocavirus (HBoV) 1 is considered an important respiratory pathogen, while the role of HBoV2-4 in clinical disease remains somewhat controversial. Since, they are characterized by a rapid evolution, worldwide surveillance of HBoVs’ genetics is necessary. This study explored the prevalence of HBoV genotypes in pediatric patients with respiratory tract infection in Croatia and studied their phylogeny. Using multiplex PCR for 15 respiratory viruses, we investigated 957 respiratory samples of children up to 18 years of age with respiratory tract infection obtained from May 2017 to March 2021 at two different hospitals in Croatia. Amplification of HBoV near-complete genome or three overlapping fragments was performed, sequenced, and their phylogenetic inferences constructed. HBoV was detected in 7.6% children with a median age of 1.36 years. Co-infection was observed in 82.2% samples. Sequencing was successfully performed on 29 HBoV positive samples, and all belonged to HBoV1. Croatian HBoV1 sequences are closely related to strains isolated worldwide, and no phylogenetic grouping based on mono- or co-infection cases or year of isolation was observed. Calculated rates of evolution for HBoV1 were 10−4 and 10−5 substitutions per site and year. Recombination was not detected among sequences from this study.

Objective: Nontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait. Materials and Methods: DNA was extracted from MGIT cultures (n = 1,033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1,033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers. Results: mPCR identified 979 isolates as MTB, 53 as NTM and 1 isolate as mixed culture. LiPA and/or PCR sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in one culture and 5 isolates only at genus level. PCR sequencing yielded more specific identification for 22 isolates at the species/subspecies level. Conclusions: mPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at the species/species complex level and 2 mixed cultures. PCR sequencing yielded more specific identification at the species/subspecies level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR sequencing is suitable for the proper management of mycobacterial infections in Kuwait.

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Made available in DSpace on 2016-08-03T15:45:32Z (GMT). No. of bitstreams: 1 2010 - Raquel Silva Lisboa.pdf: 4230911 bytes, checksum: e7087ccd29d417f592ca09ec5f49c657 (MD5) Previous issue date: 2010-04-14
Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, CNPq.
Dogs can be infected with various hemoparasites, and the occurrence of co-infections between Ehrlichia canis, Babesia canis, Anaplasma platys, and Hepatozoon canis species is very common, since they have the same tick vector. The objectives of this study were to delineate a multiplex PCR technique for the simultaneous diagnostic of microorganisms of Babesia and Ehrlichia genera in canine blood samples, and to realize the partial characterization of fragments of the 16S rRNA gene of the family Anaplasmataceae agents and, of 18S rRNA gene of Babesia detected in some samples PCR-positive, comparing the sequences obtained with sequences of other strains previously deposited in GenBank. Total DNA of 119 blood samples was extracted, of these, 40 were selected by showing cytoplasmatic inclusions in leukocytes and/or platelets suggesting infection by agents of Anaplasmataceae family (1E to 40E), 37 by showing piroplasms (1B to 37B), and two by presenting structures of both agents (M1 and M2), and finally, 40 samples with negative parasitological diagnostic and hematological exam without alterations. All these samples were tested by PCR to confirm the absence or presence of these hemoparasites, and them utilized in the multiplex PCR delineation. In multiplex PCR reactions the primers A17/EC3 were used to amplify an approximately 600bp region of the 16S rRNA gene of Ehrlichia species and the primers PIRO-A1/PIRO-B were used to amplify an approximately 450bp region of the 18S rRNA gene of Babesia species. Validation of multiplex PCR was performed by real time multiplex PCR. The multiplex PCR was able to simultaneously detect both agents in a DNA sample of a dog naturally co-infected and all the single infections by Babesia, but does not detected all the Ehrlichia infections. The real-time multiplex PCR was more sensitive in detect both single and also co-infections, as well as positive DNA mixtures for the two agents. The sequencing results confirmed the isolates identity, and that the primers PIRO-A1/PIRO-B also amplified the DNA of Hepatozoon canis. Phylogenetic analysis indicated that E. canis, A. platys, B. canis and H. canis species found in this study showed close similarities with sequences previously deposited in GenBank, forming monophyletic groups.
Os c?es podem se infectar com diversos hemoparasitos, sendo muito comum a ocorr?ncia de coinfec??es entre as esp?cies Ehrlichia canis, Babesia canis, Anaplasma platys e Hepatozoon canis, visto que possuem o mesmo carrapato vetor. Este estudo teve como objetivos delinear uma t?cnica de PCR multiplex para diagnosticar simultaneamente microrganismos dos g?neros Ehrlichia e Babesia em amostras de sangue de c?es e realizar a caracteriza??o parcial de fragmentos do gene 16S rRNA de agentes da fam?lia Anaplasmataceae e do gene 18S rRNA de Babesia detectados em algumas amostras positivas pela PCR, comparando as sequ?ncias obtidas com as sequ?ncias de outras cepas depositadas previamente no GenBank. O DNA total de 119 amostras de sangue foi extra?do. Destas, 40 foram selecionadas por apresentar inclus?es citoplasm?ticas em leuc?citos e/ou plaquetas sugestivas de infec??o por agentes da fam?lia Anaplasmataceae (1E a 40E), 37 por apresentar formas parasit?rias de piroplasm?deos (1B a 37B), duas por apresentar estruturas de ambos os agentes (M1 e M2) e, finalmente, 40 amostras com diagn?stico parasitol?gico negativo e exame hematol?gico sem altera??es. Todas estas amostras foram testadas por PCR, para a confirma??o da aus?ncia ou presen?a destes hemoparasitos, e depois utilizadas no delineamento da PCR multiplex. Nas rea??es de PCR multiplex utilizou-se os oligonucleot?deos iniciadores A17/EC3 que amplificam um produto de aproximadamente 600pb de uma por??o do gene 16S rRNA de esp?cies de Ehrlichia e os oligonucleot?deos iniciadores PIRO-A1/PIRO-B que amplificam um produto de aproximadamente 450pb de uma por??o do gene 18Sr RNA de esp?cies de Babesia. A valida??o da PCR multiplex foi realizada por PCR multiplex em tempo-real. A PCR multiplex foi capaz de detectar simultaneamente os dois agentes em uma amostra de DNA de um c?o naturalmente coinfectado e todas as infec??es individuais por Babesia, mas n?o detectou todas as infec??es por Ehrlichia. A PCR multiplex em tempo real foi mais sens?vel em detectar tanto infec??es ?nicas quanto coinfec??es, al?m de misturas de DNA positivo para os dois agentes. Os resultados dos sequenciamentos confirmaram a identidade dos isolados, e que os oligonucleot?deos PIRO-A1/PIRO-B amplificaram tamb?m, o DNA de Hepatozoon canis. As an?lises filogen?ticas indicaram que as esp?cies de E. canis, A. platys, B. canis e H. canis encontradas neste estudo possuem similaridades pr?ximas com sequ?ncias previamente depositadas no GenBank, formando grupos monofil?ticos.

Abstract The goal of phylogenetics is to construct relationships that are true representations of the evolutionary history of a group of organisms or genes. The history inferred from phylogenetic analysis is usually depicted as branching in tree-like diagrams or networks. In nematology, phylogenetic studies have been applied to resolve a wide range of questions dealing with improving classifications and testing evolution processes, such as co-evolution, biogeography and many others. There are several main steps involved in a phylogenetic study: (i) selection of ingroup and outgroup taxa for a study; (ii) selection of one or several gene fragments for a study; (iii) sample collection, obtaining PCR products and sequencing of gene fragments; (iv) visualization, editing raw sequence data and sequence assembling; (v) search for sequence similarity in a public database; (vi) making and editing multiple alignment of sequences; (vii) selecting appropriate DNA model for a dataset; (viii) phylogenetic reconstruction using minimum evolution, maximum parsimony, maximum likelihood and Bayesian inference; (ix) visualization of tree files and preparation of tree for a publication; and (x) sequence submission to a public database. Molecular phylogenetic study requires particularly careful planning because it is usually relatively expensive in terms of the cost in reagents and time.

Abstract The goal of phylogenetics is to construct relationships that are true representations of the evolutionary history of a group of organisms or genes. The history inferred from phylogenetic analysis is usually depicted as branching in tree-like diagrams or networks. In nematology, phylogenetic studies have been applied to resolve a wide range of questions dealing with improving classifications and testing evolution processes, such as co-evolution, biogeography and many others. There are several main steps involved in a phylogenetic study: (i) selection of ingroup and outgroup taxa for a study; (ii) selection of one or several gene fragments for a study; (iii) sample collection, obtaining PCR products and sequencing of gene fragments; (iv) visualization, editing raw sequence data and sequence assembling; (v) search for sequence similarity in a public database; (vi) making and editing multiple alignment of sequences; (vii) selecting appropriate DNA model for a dataset; (viii) phylogenetic reconstruction using minimum evolution, maximum parsimony, maximum likelihood and Bayesian inference; (ix) visualization of tree files and preparation of tree for a publication; and (x) sequence submission to a public database. Molecular phylogenetic study requires particularly careful planning because it is usually relatively expensive in terms of the cost in reagents and time.