Polymerase Chain Reaction Products Research Articles

Correlation of rs35753505 polymorphism in Neuregulin 1 gene with psychopathology and intelligence of people with schizophrenia

Background and aimSchizophrenia is one of the most severe psychiatric disorders. About 0.5 to 1% of the world's population suffers from this non-Mendelian disorder. Environmental and genetic factors seem to be involved in this disorder. In this article, we investigate the alleles and genotypic correlation of mononucleotide rs35753505 polymorphism of Neuregulin 1 (NRG1), one of the selected genes of schizophrenia, with psychopathology and intelligence. Materials and methods102 independent and 98 healthy patients participated in this study. DNA was extracted by the salting out method and the polymorphism (rs35753505) were amplified by polymerase chain reaction (PCR). Sanger sequencing was performed on PCR products. Allele frequency analysis was performed using COCAPHASE software, and genotype analysis was performed using Clump22 software. ResultsAccording to our study's statistical findings, all case samples from the three categories of men, women, and overall participants significantly differed from the control group in terms of the prevalence of allele C and the CC risk genotype. The rs35753505 polymorphism significantly raised Positive and Negative Syndrome Scale (PANSS) test results, according to a correlation analysis between the two variables. However, this polymorphism led to a significant decrease in overall intelligence in case samples compared to control samples. ConclusionIn this study, it seems that the rs35753505 polymorphism of NRG1 gene has a significant role in the sample of patients with schizophrenia in Iran and also in psychopathology and intelligence disorders.

Liver Fluke Species Identification Isolated From Humans and Animals Using PCR-RFLP and DNA Sequencing

Fasciola species are a member of flatworms belonging to the trematodes (flukes), commonly known as liver fluke, they are extremely pathogenic parasites that affect the liver of humans and animals, nowadays, most laboratories and research facilities use molecular-based techniques for identifying and describing Fasciola species. The molecular diagnostic markers such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and PCR-RFLP methods are accurate and more specific than the immunological and microscopical methods. The identification of the species of liver flukes will give a new clue for the treatment and control of fascioliasis. The aim, of this study, is to find the molecular characterization of Fasciola spp. isolated from humans and animals in Sulaimani city. The flukes were isolated from humans using endoscopic techniques and from slaughtered livestock at the new slaughterhouse of Sulaimani, 48 liver flukes were collected from different hosts; human (n = 3), cattle (n = 20), sheep (n = 20), and goats (n = 5) from October 2021 to April 2022. The uinversal primers ribosomal Deoxy Ribo Nuclic Acid (rDNA) were used, then the PCR products were subjected to restriction fragment polymorphism (RFLP) assay and The PCR Product was digested with restriction enzymes DraII, also the DNA sequencing was used for the PCR product of the primer Cytochrome Oxidase subuint 1 (COX1). The results of the PCR-RFLP of the 28s rDNA show the genetic polymorphisms among the flukes and two patterns of RFLP were observed F. hepatica, and F. gigantica, also the sequence analysis of the partial gene of the COX1 showed the isolated flukes belonged to F. hepatica and F. gigantica with some genetic variation, and the result of the sequences was deposited in the Gene Bank under the following Accession numbers; F. gigantica (OP718780 and OP718781) and F. hepatica (OP718782, OP718783, and OP718784). The present study concludes thatF. hepatica and F. gigantica are both responsible for human and animal Fasciolasis in Kurdistan-Iraq, Therefore, RFLP techniques and DNA sequencing are a reliable, and differential method for species and genotyping identification of liver fluke.

Vitamin D Receptor Polymorphisms Among the Turkish Population are Associated with Multiple Sclerosis.

Multiple sclerosis (MS) is an inflammatory disease characterized by demyelination and axonal degeneration affecting the central nervous system. Among the genetic factors suggested to be associated with this disease are polymorphisms to the vitamin D receptor (VDR) gene. We tested the hypothesis that polymorphisms in the vitamin D receptor (VDR) gene are associated with MS. The aim of the study was to investigate the relationship of MS with the VDR gene Fok-I, Bsm-I and Taq-I polymorphisms among the Turkish population. This study contains 271 MS patients and 203 healthy controls. Genomic DNA was isolated from the samples and the VDR gene Fok-I, Bsm-I and Taq-I polymorphism regions were amplified by polymerase chain reaction (PCR). The PCR products were digested, and the genotypes were determined based on size of digested PCR products. Our results demonstrate associations between MS and the distribution of the VDR gene Fok-I T/T polymorphism genotype in a dominant model, VDR gene Fok-I T allele frequency, distribution of VDR gene Taq-I C/C polymorphism genotype in a dominant model and VDR gene Taq-I C allele frequency (Pearson test, p<0.05). However, there was no association between MS and the VDR gene Bsm-I polymorphisms for the genotype distribution (Pearson test, p>0.05) or allele frequency (Pearson test, p>0.05). Fok-I and Taq-I VDR gene polymorphisms are significantly associated with MS in dominant, homozygote and heterozygote inheritance models among the Turkish population.

Frequency and molecular basis of CD36 deficiency among platelet donors in Kunming, China

CD36 is a multifunctional receptor expressed on the surface of many cell types. Among healthy individuals, CD36 may be absent on platelets and monocytes (type I deficiency) or platelets alone (type II deficiency). However, the exact molecular mechanisms underlying CD36 deficiency remain unclear. In this study, we aimed to identify individuals with CD36 deficiency and investigate the molecular basis underlying it. Blood samples were collected from platelet donors at Kunming Blood Center. Platelets and monocytes were isolated and CD36-expression levels were analyzed using flow cytometry. DNA from whole blood and mRNA isolated from monocytes and platelets of individuals with CD36 deficiency were analyzed using polymerase chain reaction (PCR) testing. The PCR products were cloned and sequenced. Among the 418 blood donors,7 (1.68%) were CD36 deficient: 1 (0.24%) with type I deficiency and 6(1.44%) with type II deficiency. Six heterozygous mutations occurred, including c.268C>T (in type I individuals), c.120 + 1 G>T, c.268C>T, c.329_330del/AC, c.1156 C>T, c.1163A>C, and c.1228_1239del/ATTGTGCCTATT (in type II individuals). Mutations were not detected in one type II individual . At the cDNA level, only mutant, but not wild-type, transcripts were detected in the platelets and monocytes of type I individual. In type II individuals, only mutant transcripts were found in platelets, whereas monocytes possessed wild-type and mutant transcripts. Interestingly, only alternative splicing transcripts were observed in the individual without mutation. We report the incidence rates of type I and II CD36 deficiencies among platelet donors in Kunming. Molecular genetic analyses of DNA and cDNA demonstrated that homozygous mutations on the cDNA level in platelets and monocytes or platelets alone identified type I and II deficiencies, respectively. Furthermore, alternatively spliced products also potentially contribute to the mechanism of CD36 deficiency.

Association of MT-ATP6 gene mutations with the pathogenesis of chronic kidney disease in human

BackgroundChronic kidney disease (CKD) is one of the major global health problems. CKD patients are at increased risk of cardiovascular diseases, anemia, mineral, and bone disorders, decreased quality of life, and early death. Involvement of mitochondria is increasingly recognized in genetic and acquired CKD. Mitochondrial dysfunctions are caused by many pathogenic mutations localized in the mitochondrial gene (MT-ATP6) that encodes the subunit ‘a’ of ATP synthase. ObjectivesThe aim of this study was to identify the MT-ATP6 gene mutations in patients with CKD and assess the relationship between MT-ATP6 gene mutation with CKD. Materials and methodsIn this study, blood samples of 18 CKD patients and 21 healthy controls were analyzed. Genomic DNA was extracted from the collected blood samples; the MT-ATP6 region was amplified by polymerase chain reaction (PCR); PCR products was purified; and direct sequencing was performed. In addition, sequencing data was further analyzed with various in silico tools. ResultsIn this study it was found that the serum creatinine concentration of CKD patients was approximately 7.5 folds higher than that of healthy controls. According to the criteria based on the estimated glomerular filtration rate (eGFR), it was determined that 78% of the patients were in stage-5 (eGFR <15 mL/min/1.73 m2) and the rest of the patients were in stage-4 (eGFR between 15 and 29 mL/min/1.73 m2) of CKD. Analyzing MT-ATP6 gene sequences of CKD patients, eight novel mutations (m.8732C>G, m.8800_8801insG, m.8804C>G, m.8947C>G, m.8983C>G, m.8999T>G, m.9059C>G, and m.9129C>G) along with 11 reported mutations were identified. In the case of CKD patients, 14 nonsynonymous mutations in the MT-ATP6 gene were detected and 8 of them were damaging and disease-causing based on in silico analyses with five different tools. On the other hand, only 3 nonsynonymous mutations were identified in healthy individuals, which were all neutral and tolerating mutations according to in silico analyses. In this study, a significant correlation between non-synonymous MT-ATP6 gene mutations and occurrence of CKD (p-value <0.0001) was also observed. ConclusionsThe results revealed that there is a link between MT-ATP6 gene mutation and the development of CKD. It can be concluded that mutations in the MT-ATP6 gene may have a role in CKD development.

The Surveillance of Borrelia Species in Camelus dromedarius and Associated Ticks: The First Detection of Borrelia miyamotoi in Egypt

A total of 133 blood samples and 1596 adult hard ticks were collected from Camelus dromedaries at Cairo and Giza slaughterhouses in Egypt. Tick species were identified by examining their morphology and sequencing the cytochrome C oxidase subunit 1 (cox1) gene. Borrelia spp. was detected using nested PCR on the IGS (16S-23S) gene, and positive samples were genotyped using 16S rRNA and glpQ spp. genes specific for Borrelia burgdorferi and Borrelia miyamotoi, respectively. The positive PCR products were sequenced and analyzed by phylogenetic tree. Analysis of the cox1 gene sequence revealed that the adult ticks belonged to three genera; Hyalomma (H), Amblyomma (Am), and Rhipicephalus (R), as well as 12 species, including H. dromedarii, H. marginatum, H. excavatum, H. anatolicum, R. annulatus, R. pulchellus, Am. testudinarium, Am. hebraeum, Am. lipidium, Am. variegatum, Am. cohaerens and Am. gemma. Borrelia spp. was found in 8.3% (11/133) of the camel blood samples and 1.3% (21/1596) of the ticks, respectively. Sequencing of the IGS (16S-23S) gene found that B. afzelii, detected from H. dromedarii and H. marginatum, and B. crocidurae, which belongs to the RF group, was detected from one blood sample. B. burgdorferi and B. miyamotoi were discovered in the blood samples and tick species. Phylogenetic analysis of the glpQ gene showed that the B. miyamotoi in this study was of the Asian and European types. These results suggest that the camels can be infected by Lyme borrelia and other Borrelia bacteria species. This study also provides the first insight into the presence of Borrelia miyamotoi and B. afzelii DNA in camels and associated ticks in Egypt.

Genetic screening of Filipinos suspected with familial Parkinson's disease: A pilot study

IntroductionAlthough genetic factors are known to play a role in the pathogenesis of Parkinson's disease (PD), true prevalence of familial PD is unknown. We conducted this pilot study to identify genes implicated in familial Parkinson's disease among Filipinos. MethodsEighteen Filipino patients belonging to 11 families with personal and family history of PD underwent thorough evaluation by movement disorders specialists. Samples were analyzed in Juntendo University, Tokyo, Japan. Sanger sequencing of polymerase chain reaction products was performed. Each sample was screened for 23 genes (SNCA, PARK 2, UCHL1, PINK 1, DJ-1, LRRK2, ATP13A2, GIGYF2, HTRA2, PLA266, FBX07, VPS35, EIF461, DNAJC13, CHCHD2, GCH1, MAPT, NR4A2, VPS13c, PSEN1, and GRN). ResultsOut of 18 patients, six harbored Parkinson-related gene mutations. Five individuals from three families were positive for PINK1 c.10140T > C(p.L347P) mutation while one had heterozygous variant PRKN c.136G>T(p.A465) gene mutation. Three families displayed autosomal recessive pattern while one family with PINK1 mutation showed autosomal dominant mode of inheritance. Bradykinesia and tremor were predominant symptoms. Mean age at onset of symptoms was 40.4 years among those with PINK1 mutations. ConclusionIn this study, we presented the clinical profiles and identified two genetic mutations among a small group of Filipino patients with familial PD. They were congruent with most studies showing these mutations as the most common causes of autosomal recessive early-onset PD. Preliminary data from this pilot study will guide planning for larger scale studies, such as collaborative projects including The Global Parkinson's Genetics Program (GP2).

Study of bacteria Listeria monocytogenes in spontaneous aborted women in Salah Al-deen province

This study was performed between January 2013 to the end of June 2013, involving 94 spontaneous aborted women at 15-45 years who attended TGH in Salah Al-deen province. They were examined and taking placental pits and cervical swabs. For diagnosis, used Gram staining, cultures, biochemical tests, API system and molecular technique including polymerase chain reaction (PCR).&#x0D; The spontaneous aborted women with Listeria infection was 13 (13.82%) out of 94 aborted women, while 81(81.86%) of aborted women had other causes of abortion. The most affected age group with Listeria infection was 25 – 34 years old represented 7 (53.84%) aborted women. The most infected patients from rural area was 61.53%, while in urban was 38.46%. The most aborted women with Listeria infection were had previous abortions within first trimester (less than 12 weeks of gestational age) were 8 (100%) cases. The pathogens isolated from placenta pits and cervical swabs was Listeria monocytogenes represented 11(37.93%) out of 29 isolated pathogens, while Listeria ivanovii was 2 (6.80%). PCR was used for detection of virulence associated gene of Listeria monocytogenes including hly A which had been found to be amplifying of their DNA fragments. The PCR products showed the eleven isolates of Listeria monocytogenes had the hly A gene

First report of maize rayado fino virus in corn fields in Santa Catarina, Brazil.

Maize (Zea mays L.) is the main cereal food of humans and animals in Brazil. In 2020 and 2021, a severe infestation of corn leafhoppers (Dalbulus maidis; Hemiptera: Cicadellidae) was observed in Santa Catarina State (South of Brazil). Subsequently, symptoms of chlorotic stripes limited in leaf veins started to appear in maize plants. Given the similarity of symptoms and the presence of high populations of corn leafhoppers in corn production areas, 30 plants in reproductive stage showing systemic symptoms were collected in summer and autumn from commercial fields of five municipalities in Santa Catarina: Campos Novos (27°23'18.0"S, 51°12'52.7"W), Lages (27°47'17.8"S, 50°18'16.9"W), Mafra (26°06'42"S, 49°48'25"W), Fraiburgo (27°01'36"S, 50°55'19"W), and Abelardo Luz (26°34'02"S, 52°20'02"W). The young leaves of these samples were used for molecular analyses targeting the maize rayado fino virus (MRFV; Tymoviridae: Marafivirus). Total nucleic acids were extracted using TRIzol® (Invitrogen, USA), following the manufacturer's instructions. These were used as a template for cDNA synthesis with the enzyme MMLV-RT (Promega, USA), following the manufacturer's instructions. The polymerase chain reaction (PCR) was performed using Gotaq® DNA polymerase (Promega, USA) and MRFV-09/MRFV-10 primers (Hammond et al. 1997). All PCR products were subjected to electrophoresis in 1% agarose gel and were visualized under ultraviolet light. Twenty-eight of the 30 tested plants were MRFV-positive, showing a fragment with an expected size of ~633 bp. To confirm our results, all MRFV-positive samples were sent for sequencing (GenBank accession numbers OM763708 - OM763710 and ON730784 - ON730806) and submitted to BLASTn search (https://blast.ncbi.nlm.nih.gov/Blast.cgi), resulting in identities ranging from 96.21% to 99.21% with the isolate "Brazil 26" of MRFV, which was detected in 2005 in São Paulo, Brazil (GenBank accession nº: AF186178) (Hammond and Bedendo 2005). A second set of primers was used to validate the first PCR, confirming MRFV infection (data not shown).Moreover, whitish streaks and leaf reddening were observed on the leaves of some plants; therefore, the identification for phytoplasmas (Candidatus Phytoplasma asteris) and spiroplasmas (Spiroplasma kunkelii) from the corn stunt complex was performed. For this, previously extracted nucleic acids from each sample were used as templates for a multiplex PCR using the primers CSSR6/CSSF2 and R16F2n/R16R2 (Gundersen and Lee 1996; Barros et al. 2001). Two plants were infected with only spiroplasma, 17 samples were infected with Spiroplasma and MRFV, and three samples were infected by these three pathogens. An increasing incidence of corn stunt has been observed in commercial fields in Santa Catarina in recent years. Mollicutes are commonly found and mostly studied as causal agents of corn stunt disease. On the contrary, despite being present in Brazil since the 1970s, the virus is less studied because its contribution to the corn stunt complex is still unknown (Hammond and Bedendo 2001). In this report, indications that the virus is expanding to different regions in southern Brazil were observed, which raises an opportunity for further evaluation and its consideration in monitoring programs. Moreover, to the best of our knowledge, this is the first report of MRFV in Santa Catarina, Brazil.

Identification and Characteristics of Multidrug-Resistant Ureaplasm urealyticum and Mycoplasma hominis Isolates among Female Patients with Recurrent Non-Gonococcal Urethritis in a Tertiary Hospital, China

Background: Concerns are increasing over the mechanisms of drug resistance pathogens among non-gonococcal urethritis (NGU) or cervicitis. The study aims to explore the distribution of Ureaplasm urealyticum (U. urealyticum) and Mycoplasma homins (M. homins) isolates, characteristics of multidrug resistance, and the underlying mechanism to allow clinicians to deliver rational therapy for NGU. Methods: Samples from cervical secretions of 246 patients with NGU were collected. Mycoplasma culture and drug susceptibility tests were performed, respectively. The resistance genes were detected by multiplex Polymerase Chain Reaction (PCR), and the PCR products were cloned and sequenced. Results: Out of 246 samples, the overall prevalence of U. urealyticum and/or M. hominis infection was 39.02% (96/246). In 96 culture positive samples, the overall infection rate of U. urealyticum was higher than that of the single M. hominis (46.87% vs. 28.13%) (p &lt; 0.05), and the coinfection rate was 25.00% (24/96). Each U. urealyticum and M. hominis isolate exhibited resistance to at least three types of antimicrobial agents (manifest by more than 70% resistance to erythromycin, tetracycline, ciprofloxacin, and spiramycin, followed by ofloxacin, roxithromycin, azithromycin and josamycine (with resistance thereto of more than 50%)). More than 80% of U. urealyticum and M. hominis isolates were susceptible to minomycine and doxycycline, whereas 81.16% and 77.44% of both U. urealyticum and M. hominis isolates were also susceptible to minomycine and doxycycline. Surprisingly, the resistance rate of the mixed infection was higher that of erythromycin, tetracycline and ciprofloxacin comparison to the single infection (p &lt; 0.05). All tetracycline-resistant isolates carried the tetM gene and 50% of erythromycin-resistant isolates carried the ermA gene. Conclusions: Among outpatients with recurrent NGU, the U. urealyticum infection dominated, followed by M. hominis, mixed U. urealyticum and M. hominis infection. Minomycine and doxycycline are recommended for empirical clinical treatment. The determination of U. urealyticum and M. hominis infection, antibiotic susceptibility testing is crucial for effective therapy.

Genetic polymorphism of prolactin and nitric oxide synthase in Holstein cattle.

Bacterial and viral infections affect the welfare of animals and lead to large economic losses in dairy cattle breeding due to decreased productive indicators and increased culling rates. In modern dairy farming, farmers are looking for effective solutions to prevent and minimize infectious disease risks. To this end, the most relevant study field is the search for gene sites that impact production and health. This study aimed to determine the nature of the distribution of the relative frequencies of alleles and genotypes of polymorphic prolactin (PRL) and nitric oxide synthase (NOS2) in Holstein cows and identify the relationship of these genes with resistance to mastitis and bovine leukemia. For this study, we chose cows because infectious diseases affect the amount of lactation and milk quality. Holstein cattle with mastitis and bovine leukemia were selected. Animal genotypes were determined by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products. The results were analyzed using a nonparametric statistical method using Microsoft Excel 2010 and Statistica 6.0. In healthy animals, 94 genotypes were identified for both genes under study. For bPRL, bPRL-RsaIAA (72) was the most common genotype and bPRL-RsaIBB (4) the least; for NOS2, bNOS2 -HinfIAB (47) was the most common genotype and bNOS2 -HinfIAA the least (21). In animals with leukemia, 34 genotypes were identified. For PRL, bPRL-RsaIAA (25) was the most common genotype and bPRL-RsaIBB (2) the least; for NOS2, bNOS2 -HinfIBB (17) was the most common genotype and bNOS2 -HinfIAA (3) the least. In animals with mastitis, 67 genotypes were identified. For PRL, bPRL-RsaIAA (43) was the most common genotype and bPRL-RsaIBB (6) the least; for NOS2, bNOS2 -HinfIBB (31) was the most common genotype and bNOS2-HinfIAA (7) the least. The distribution of genotypes of polymorphic bPRL and bNOS2 generally coincides, and bPRL-RsaIBB is the most common genotype. In groups of sick animals, the number of bNOS2 -HinfIAA homozygotes was lower than that of the control group. In particular, the proportion of animals with the bNOS2 -HinfIAA genotype with bovine leukemia was 8.7% and with mastitis was 10.3% compared with 22.4% in healthy animals. These data support the possible association of the bNOS2 -HinfIAA genotype with resistance to infection. The frequency of the bPRL-RsaIB allele was higher in groups of sick animals. This allele is associated with increased milk productivity, suggesting that highly productive animals are less resistant to the incidence of viral bovine leukemia and mastitis of bacterial etiology. DNA amplification of Holstein cattle for the polymorphic regions of PRL and NOS2 using the PCR-RFLP method revealed a possible connection between the distribution of relative allele frequencies of bPRL and bNOS2 and resistance to viral and bacterial infections. Thus, in groups of sick animals, the frequency of bPRL-RsaIBB, associated with increased milk production compared with the theoretically calculated equilibrium value was higher and the number of homozygotes bNOS2 -HinfIAA was lower than in the control group. In conclusion, animals with increased milk production were more prone to diseases, such as mastitis and bovine leukemia.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

Simple visualization method for the c.577del of erythropoietin variant: CRISPR/dCas9-based single nucleotide polymorphism diagnosis.

One of the single nucleotide polymorphisms (SNPs) in human erythropoietin (hEPO), the c.577del variant, can produces 26 amino acids longer than the wild-type hEPO, posing a risk of misinterpretation in routine doping analysis. To prevent this, the World Anti-Doping Agency (WADA) included a procedure for reporting the sequencing results regarding the presence or absence of SNPs for suspected cases in the new version of the technical document for recombinant EPO in 2022. However, it is very expensive for anti-doping laboratories to purchase a gene sequencing analyzer, which costs hundreds of thousands of dollars, and only a few companies provide specific gene sequencing services with accredited certification. Therefore, in this study, we developed a simple visualization method for the c.577del of the EPO variant at the gene level. The gene fragment of the EPO gene, including c.577del, was amplified using a fast polymerase chain reaction (PCR), and the PCR products were incubated with the clustered regularly interspaced short palindromic repeats (CRISPR)/deadCas9 system using variant-specific single-guide RNA (sgRNA). This ribonucleoprotein complex binds specifically to the EPO variant gene fragment, causing a band shift on native-PAGE. We designed 4 sgRNAs that can bind only to the EPO variant or wild-type gene. In addition, an electrophoretic mobility shift assay on a gel demonstrated that one of the sgRNAs had a high level of specificity. Consequently, the c.577del variant was selectively detected by visualizing the target fragment of the gene on the gel within 3h using only 3μl of the whole blood.

Detection of Tobacco Bacterial Wilt Caused by Ralstonia solanacearum by Combining Polymerase Chain Reaction with an α-Hemolysin Nanopore.

Tobacco bacterial wilt is a serious disease caused by the soil-borne bacterium Ralstonia solanacearum (R. solanacearum). Herein, a rapid and purification-free α-hemolysin (α-HL) nanopore-sensing strategy based on polymerase chain reaction (PCR) and lambda exonuclease digestion was established to detect R. solanacearum. A 198-nucleotide-long single-stranded DNA was obtained via asymmetric PCR or the lambda exonuclease-mediated digestion of the PCR product. The DNA fragment produced unique long-lived, current-blocking signals when it passed through the α-HL nanopore. This sensing approach can allow for the determination of R. solanacearum in tobacco samples and can be conveniently extended to other DNA monitoring because of the extremely wide range of PCR applications.

Identification of Cyanobacteria and Its Potential Toxins in the Joanes I Reservoir, Bahia, Brazil

The Joanes I Reservoir is responsible for 40% of the drinking water supply of the Metropolitan Region of Salvador, Bahia, Brazil. For water sources such as this, there is concern regarding the proliferation of potentially toxin-producing cyanobacteria, which can cause environmental and public health impacts. To evaluate the presence of cyanobacteria and their cyanotoxins in the water of this reservoir, the cyanobacteria were identified by microscopy; the presence of the genes of the cyanotoxin-producing cyanobacteria was detected by molecular methods (polymerase chain reaction (PCR)/sequencing); and the presence of toxins was determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The water samples were collected at four sampling points in the Joanes I Reservoir in a monitoring campaign conducted during the occurrence of phytoplankton blooms, and the water quality parameters were also analysed. Ten cyanobacteria species/genera were identified at the monitoring sites, including five potentially cyanotoxin-producing species, such as Cylindrospermopsis raciborskii, Cylindrospermopsis cf. acuminato-crispa, Aphanocapsa sp., Phormidium sp., and Pseudanabaena sp. A positive result for the presence of the cylindrospermopsin toxin was confirmed at two sampling points by LC-MS/MS, which indicated that the populations are actively producing toxins. The analysis of the PCR products using the HEPF/HEPR primer pair for the detection of the microcystin biosynthesis gene mcyE was positive for the analysed samples. The results of this study point to the worrisome condition of this reservoir, from which water is collected for public supply, and indicate the importance of the joint use of different methods for the analysis of cyanobacteria and their toxins in reservoir monitoring.

Low Throughput Direct Cycle Sequencing of Polymerase Chain Reaction (PCR) Products.

Polymerase Chain Reaction (PCR) products have been traditionally characterized by cloning and cycle sequencing. However, when quick sequencing data are required, the cloning step may be omitted and PCR products can be sequenced directly. We describe here a sequencing protocol that involves the gold standard Big Dye chemistry in a low throughput format using one of the latest sequencing platforms the ABI Seqstudio. Our cycle sequencing protocol follows the following steps: (1) purification of the PCR product with a spin column-based kit; (2) quality & quantity assessment of the PCR product with the use of spectrophotometry & gel electrophoresis; (3) setup and amplification of the cycle sequencing reaction; (4) Capillary Electrophoresis; (5) Sequence Data Analysis.

Gold Nanoparticle and Polymerase Chain Reaction (PCR)-Based Colorimetric Assay for the Identification of Campylobacter spp. in Chicken Carcass.

Campylobacteriosis is a common cause of gastrointestinal disease. In this study, we suggest a general strategy of applying gold nanoparticles (AuNPs) in colorimetric biosensors to detect Campylobacter in chicken carcass. Polymerase chain reaction (PCR) was utilized for the amplification of the target genes, and the thiolated PCR products were collected. Following the blending of colloid AuNPs with PCR products, the thiol bound to the surface of AuNPs, forming AuNP-PCR products. The PCR products had a sufficient negative charge, which enabled AuNPs to maintain a dispersed formation under electrostatic repulsion. This platform presented a color change as AuNPs aggregate. It did not need additional time and optimization of pH for PCR amplicons to adhere to the AuNPs. The specificity of AuNPs of modified primer pairs for mapA from Campylobacter jejuni and ceuE from Campylobacter coli was activated perfectly (C. jejuni, p-value: 0.0085; C. coli, p-value: 0.0239) when compared to Salmonella Enteritidis and Escherichia coli as non-Campylobacter species. Likewise, C. jejuni was successfully detected from artificially contaminated chicken carcass samples. According to the sensitivity test, at least 15 ng/μL of Campylobacter PCR products or 1×103 CFU/mL of cells in the broth was needed for the detection using the optical method.

Molecular characterization of Streptococcus agalactiae and Streptococcus dysgalactiae causing bovine mastitis in the southern region of Bangladesh.

This study was conducted to validate polymerase chain reaction (PCR) as a confirmatory diagnostic tool to find out the presence and frequency of Streptococcus agalactiae (S. agalactiae) and Streptococcus dysgalactiae (S. dysgalactiae) in mastitic milk samples obtained from dairy cows in the southern region of Bangladesh. A total of 196 samples of bovine milk were collected from various dairy farms in the Chattogram metropolitan area of the southern part of Bangladesh. DNA extracted from isolates obtained by culturing California mastitis test (CMT)-positive mastitic milk samples (n = 146) on 5% sheep blood agar was used as a template for PCR. Two sets of specific primers based on the 16S rRNA gene were used to discriminate between S. agalactiae and S. dysgalactiae. Four PCR products were subjected to sequencing, followed by phylogenetic analysis. The PCR analyses revealed that out of the 146 CMT-positive milk samples tested, 29 samples were positive for S. agalactiae (19.86%), while 26 samples were positive for S. dysgalactiae (17.81%). Further sequence analysis of the corresponding PCR products and bioinformatics analysis verified the results. The study proves the efficiency of PCR as a useful diagnostic approach to determine the presence and prevalence of S. agalactiae and S. dysgalactiae in mastitic milk samples obtained from dairy cows.

Distribution and antibiogram of Vibrio species from hospital wastewater in Southwest, Nigeria.

the continuous generation of wastewater and its release into the environment with little or no treatment remains a threat to the environment and public health. We examined the prevalence and antibiotic susceptibility profiles of Vibrio species isolated from untreated wastewater samples from Ondo State Specialist Hospital Okitipupa, Nigeria, as part of the global efforts to provide information for containing the spread of resistant infections. twelve hospital wastewater samples were collected aseptically and transported to the laboratory for analysis. The samples were processed on thiosulphate citrate bile salt sucrose agar and colonies typical of Vibrio species were selected for further identification. All isolates were confirmed by polymerase chain reaction (PCR) using Vibrio-specific primers and the PCR products were sequenced for species identification. The susceptibility profiles of the isolates were determined by the Kirby-Bauer disc diffusion method. twenty-nine (58%) of 38 presumptive isolates were confirmed as Vibrio by PCR, while 23 (60.5%) isolates were screened up to species level by sequencing. Six different species following the trend: 26.1% V. fortis and V. algivorus, 17.4% V. cholerae, 13.0% V. panuliri, 8.7% V. stylophorae and V. parahaemolyticus were identified. The isolates were commonly resistant (73.9%-91.3%) to doxycycline, tetracycline, erythromycin and meropenem. The least resistance rate (17.4%) was observed against amikacin and cotrimoxazole. All isolates were multidrug-resistant, with multiple antibiotic resistance (MAR) indices exceeding the 0.2 recommended limit. this study has shown that untreated hospital wastewater is a reservoir for diverse strains of multiply resistant Vibrio species. Therefore, it is essential to adequately treat hospital wastewater to eliminate these emerging pollutants and set up a monitoring scheme to evaluate the treatment plants' effectiveness to reduce the pollutants' impact on the environment and the population.

Annals of Clinical and Medical Case Reports

1.1. Objective: Gitelman syndrome (GS) is an autosomal recessive tubular disorder characterized by metabolic alkalosis, hypokalemia, hypomagnesemia and hypocalciuria. GS is mostly caused by inactivating mutations of the SLC12A3 gene. The purpose of this study was to describe the clinical features of a GS patient and investigate the underlying mutations of SLC12A3 gene in the pedigree. 1.2. Methods: A patient suffering from muscle weakness was clinically diagnosed as GS. Clinical data of the proband were studied retrospectively. All of his family members were screened for SLC12A3 gene mutations. 26 exons and exon-intron boundaries of SLC12A3 gene were amplified by Polymerase Chain Reaction(PCR). PCR products were sequenced directly. 1.3. Results: The proband had hyperreninemia but hypoaldosteronemia, which was distinct from the cases previously reported. The proband and his sick brother were found to have the same compound heterozygous mutations (c.917C&gt;T and IVS 14-8T&gt;C) of SLC12A3 gene. Each mutation was detected in paternal and maternal genomic DNA, respectively. The proband’s healthy brother had one mutation (c.917C&gt;T) only. IVS 14-8T&gt;C was a novel splicing site mutation that had never been reported. 1.4. Conclusion: Hypoaldosteronemia was found in a GS patient. A novel heterozygous splicing site mutation of the SLC12A3 gene was reported, expanding the spectrum of SLC12A3 gene mutations.