Multiplex Polymerase Chain Reaction Technique Research Articles

NEW PRIMER DESIGN of MULTIPLEX POLYMERASE CHAIN REACTION (PCR) for DETECTION of Escherichia Coli and Salmonella Enterica in FOOD SAMPLE

Background: Multiplex PCR techniques are used to detect multiple pathogens at the same time simultaneously. In order to achieve the specificity and sensitivity of detection, it is important to optimize the multiplex PCR method properly. Developing suitable primers and optimizing PCR temperature to boost particular genes from pathogens are key factors for this optimization. Methods: For the simultaneous detection of Escherichia coli and Salmonella enterica in food samples, this research aims to develop a set of primers for multiplex polymerase chain reaction (PCR). Using Primer-BLAST software, the study utilizes specific primer designs for the phoA gene (Eschericia coli) and invA gene (Salmonella enterica). DNA isolation has confirmed successful extraction from the two bacterial samples. PCR was performed under different conditions, including Singleplex and Multiplex PCR, using two annealing temperatures of 53oC and 50oC. Results: The results showed that this method can effectively amplify target genes and indicate their specificity and reliability at both temperature levels. Given these results, it has successfully conducted multiplex PCR using the built primer pairs. Both annealing temperatures of 50oC and 53oC can be used to perform multiplex PCR to detect E. coli and Salmonella enterica in one PCR reaction. Conclusion: Through this research, we have created a new set of Multiplex PCR Primers and an optimized multiplex PCR technique for the simultaneous detection of E coli and Salmonella enterica in food samples. The rapid and simultaneous screening of E. coli and S. enterica, which contributes to improved food safety measures and pathogen detection in the food industry, is promising with this optimized PCR approach.

Pediatric T-cell lymphoblastic lymphomas but not leukemias harbor TRB::NOTCH1 fusions with unfavorable outcome

AbstractT-cell lymphoblastic lymphoma (T-LBL) and T-cell acute lymphoblastic leukemia (T-ALL) have common and distinguishing clinical and molecular features. Molecular prognostic factors are needed for T-LBL. We assessed the prevalence and prognostic impact of the T-cell receptor β (TRB)::NOTCH1 fusion in 192 pediatric patients with T-LBL and 167 pediatric patients with T-ALL, using novel multiplex polymerase chain reaction and genomic capture high-throughput sequencing techniques. The fusion was detected in 12 patients with T-LBL (6.3%) but in none of the patients with T-ALL (P = .0006, Fisher exact test). In T-LBL, the TRB::NOTCH1 fusion was associated with a significantly higher incidence of relapse (67% vs 17% in gene fusion-negative patients, P < .001, Fisher exact test). The breakpoint in TRB was most frequently located in J2-7 (n = 6). In NOTCH1, the breakpoints varied between exon 24 and 27. Consequently, a truncated NOTCH1 with its dimerization, regulation, and signal transduction domains gets controlled by strong TRB enhancer elements. This study reveals a novel recurrent genetic variant with significant prognostic relevance in T-LBL, which was absent in T-ALL. The TRB::NOTCH1 fusion in T-LBL suggests a possible unique pathogenic mechanism divergent from T-ALL. Further studies will validate the role of the TRB::NOTCH1 fusion as prognostic marker in T-LBL and elucidate its pathogenic mechanisms.

Optimization of Multiplex-PCR Technique To Determine Azf Deletions in infertility Male Patients.

To optimize the multiplex polymerase chain reaction (M-PCR) technique to diagnose microdeletions of azoospermia factors (AZF) on the Y chromosome and initially apply the technique to diagnose male patients with sperm density less than 5×106 million sperm/mL was assigned to do a test to check for AZF microdeletions on the Y chromosome. Based on the positive control samples which belong to male subjects who have had 2 healthy children without any assisted reproductive technologies, the M-PCR method was developed to detect simultaneously and accurately AZF microdeletions on 32 male patients with sperm densities below 5×106 million sperm/mL of semen at the Department of Biology and Medical Genetics - Vietnam Military Medical University. Successful optimization of the M-PCR technique including 7 reactions arranged according to each AZFabc region using 24 STS/gene on the Y chromosome. Initial application to diagnose AZF deletion on 32 azoospermic and oligospermic men reveals that AZFa deletion accounts for 6.25% (2/32); deletion of all 3 regions AZFa,b,c with 18.75% (6/32 cases); The combined deletion rate of AZFb,c is highest, accounting for 56.24% (18/32 patients). Successfully optimized the M-PCR technique in identifying AZF microdeletions using 24 sequence tagged sites (STS)/gene for azoospermic and oligozoospermic men. The M-PCR technique has great potential in the application of AZF deletion diagnosis.

Epidemiological Characterization of Respiratory Pathogens Using the Multiplex PCR FilmArray™ Respiratory Panel.

Various pathogens can cause upper respiratory tract infections, presenting challenges in accurate diagnosis due to similar symptomatology. Therefore, rapid and precise diagnostic tests are crucial for effective treatment planning. Traditional culture-based methods for diagnosis are limited by their reliance on skilled personnel and lengthy processing times. In contrast, multiplex polymerase chain reaction (PCR) techniques offer enhanced accuracy and speed in identifying respiratory pathogens. In this study, we aimed to assess the efficacy of the FilmArray™ Respiratory Panel (RP), a multiplex PCR test capable of simultaneously screening 20 pathogens. This retrospective analysis was conducted at Dankook University Hospital, South Korea, between January 2018 and December 2022. Samples from patients with upper respiratory tract infections were analyzed. Results revealed adenovirus as the most prevalent pathogen (18.9%), followed by influenza virus A (16.5%), among others. Notably, a 22.5% co-infection rate was observed. The FilmArray™ RP method successfully identified 20 pathogens within 2 h, facilitating prompt treatment decisions and mitigating unnecessary antibiotic prescriptions. This study underscores the utility of multiplex PCR in respiratory pathogen identification, offering valuable insights for epidemiological surveillance and diagnosis.

Bacterial Flora and Treatment Strategies in Women With Escherichia coli Urinary Tract Infections.

Introduction This study explores the intricate relationship between bacterial flora and the occurrence of Escherichia coli (E. coli) infections in gynecological patients. It aims to provide insights into the various treatment strategies used to effectively manage bacterial pathogens, especially E. coli infections. By conducting a comprehensive analysis of the bacterial flora in gynecological patients, the study highlights the notable presence of E. coli, promptingfurther investigation into the factors that contribute to its colonization. The objective of the study is to comprehensively investigate and detect urinary tract infections (UTIs) specifically caused by E. coli among gynecological patients. The study aims to delve into bacterial flora prevalence, antibiotic resistance patterns, and potential virulence factors. Through this analysis, the study intends to identify effective strategies for rapid detection and diagnosis of UTIs caused by E. coliby utilizing advanced microbiological and molecular techniques. Furthermore, the study aims to formulate and propose a strategic treatment approach with a particular emphasis on selecting appropriate antibiotics to reduce the risk of severe infections and associated complications. Materials and methods The methodology employed in this study included the isolation and characterization of bacterial strains from clinical samples obtained from gynecological patients. A total of 52 urine specimens were collected from patients with complaints of infection in the urinary tract and infertility. These samples underwent both preliminary and confirmatory microbiological analysis, such as gram staining, biochemical confirmation test, and antibiotic susceptibility, and further proceeded with the multiplex polymerase chain reaction (PCR) technique. The results of PCR and antibiotic susceptibility revealed the specific gene involvement and resistant characteristics of E. coli. Results The findings revealed a total of 32 specimens positive for E. coli, of which 10 patients had infertility complaints and 22 patients had UTIs. The preliminary test, gram staining, showed the gram-negative bacilli E. coli, and the nutrient agar plate revealed smooth circular translucent colonies; MacConkey agar showed pink-colored lactose-fermented colonies; and the blood and chocolate agar plates showed grayish white moist gamma-hemolytic colonies. The biochemical confirmation of E. coli resulted in positive for indole and methyl red tests and negative for Voges-Proskauer and citrate utilization tests. The multiplex PCR analysis confirmed the E. coli strains with the presence of two target genes,stx2d and stx2e. Conclusion To summarize, this study offers valuable insights into the bacterial flora of gynecological patients impacted by E. coli infections, which provides a foundation for the development of precise and efficient treatment strategies. The results emphasize the importance of personalized treatment approaches that consider both the microbiological characteristics of the infection and the evolving landscape of antibiotic resistance. The implication of this research extends to enhancing clinical outcomes and alleviating the burden of E. coli infections in gynecological settings.

Impact of Multiplex PCR in the Therapeutic Management of Severe Bacterial Pneumonia.

Pneumonia is a common and severe illness that requires prompt and effective management. Advanced, rapid, and accurate tools are needed to diagnose patients with severe bacterial pneumonia, and to rapidly select appropriate antimicrobial therapy, which must be initiated within the first few hours of care. Two multiplex molecular tests, Unyvero HPN and FilmArray Pneumonia+ Panel, have been developed using the multiplex polymerase chain reaction (mPCR) technique to rapidly identify pathogens and their main antibiotic resistance mechanisms from patient respiratory specimens. Performance evaluation of these tests showed strong correlations with reference techniques. However, good knowledge of their indications, targets, and limitations is essential. Collaboration with microbiologists is, therefore, crucial for their appropriate use. Under these conditions, and with standardized management, these rapid tests can improve the therapeutic management of severe pneumonia faster, more precisely, and with narrow-spectrum antibiotic therapy. Further randomized controlled trials are needed to address the many unanswered questions about multiplex rapid molecular testing during the diagnosis and the management of severe pneumonia. This narrative review will address the current knowledge, advantages, and disadvantages of these tests, and propose solutions for their routine use.

Development of a high-throughput DNA isolation method for livestock and poultry meat based on mesoporous metal-organic framework-coated solid-phase microextraction device

A rapid, portable, simple, low-cost, and high-throughput DNA isolation method was developed for the quantitative identification of meat adulteration based on mesoporous UiO-66 coated solid phase microextraction (SPME) devices. The high-throughput DNA extraction system consists of 96 UiO-66 SPME devices and a polyformaldehyde plate, which can extract DNA from 96 livestock and poultry meat samples simultaneously. At the same time, several parameters of the high throughput DNA isolation method were optimized, including lysis buffer volume, elution buffer volume, lysis time and elution time, etc. Compared with the single DNA isolation device, the 96-SPME high-throughput extraction system maintains the original good universality, reproducibility, chemical stability, and reusability. On this basis, it obtained higher DNA concentration (80 ± 34 ng μL−1), satisfactory DNA purity (A260/A280=2.4) and shorter extraction time (less than 7.2 s per sample), which are much more convenient and efficient than the conventional kit method and the single SPME DNA isolation method. In addition, the conventional multiplex polymerase chain reaction and real-time polymerase chain reaction techniques were used for the qualitative and quantitative detection of meat adulteration. The DNA obtained from this extraction method is highly reproducible and sensitive for the identification of duck ingredients in beef, with a detection range of 1–100 % and a minimum detection limit of 1 % (w/w).

Detection of mixed infections among three begomoviruses in pumpkin using multiplex polymerase chain reaction technique

Detection of mixed infections among three begomoviruses in pumpkin using multiplex polymerase chain reaction technique

Molecular Detection of Hepatits B Virus Genotypes in Tertiary Hospitals in Bayelsa State, Nigeria

In Africa, the Hepatitis B Virus (HBV) is considered endemic or even hyper-endemic. However, there is lack adequate comprehensive data regarding the genotypes of HBV and their distribution within Nigeria. This study seeks to identify the prevailing HBV genotypes among patients who sought medical care at the Federal Medical Center, Yenagoa and Niger Delta University Teaching Hospital Okolobiri, Bayelsa State, Nigeria. Between January and June 2022, 656 patients’ blood samples were collected from both hospitals for analysis, consisting of 475 females (72.4%) and 181 males (27.6%). The samples were tested for Hepatitis B surface antigen (HBsAg) and genotyping using immunochromatography and multiplex Polymerase Chain Reaction (PCR) techniques with type-specific primers. Among the 656 patients screened for HBsAg, 66 individuals (10%), comprising 36 females (5.4%) and 30 males (4.6%), tested positive using immunochromatography. These positive cases underwent molecular genotyping using specific primers for genotypes A, B, C, D, E, and F. Of these, 33 (50%) exhibited strong or active positivity. In comparison, the remaining 50% displayed passive positivity due to viral degradation, as HBV is a non-enveloped virus. The results further revealed that HBV genotypes E and B were the predominant types, with a prevalence of 82.4% and 11.8%, respectively, in the study area. Interestingly, the observation showed that co-infections involving HBV/B and HBV/E had a majority of 5.9% and were detected among two female patients aged 26 and 25. It is noteworthy that in the study area and its environs, healthcare providers commonly prescribe tenofovir, a nucleotide analog drug. Previous research by various authors has indicated that HBV genotype E is more responsive to nucleotide analogs, while HBV genotype B responds better to interferon-based therapies. In conclusion, this study highlights the prevalence of HBV genotypes B, E, and B+E coinfection in Yenagoa within the Niger Delta region of Nigeria. This knowledge underscores the importance of healthcare providers accurately diagnosing HBV genotypes before prescribing treatment regimens, particularly considering combination therapy, to optimize the care of infected patients. This approach is vital for effectively managing HBV infections and improving patient outcomes.

Molecular Identification and Detection of Streptococcus pneumoniae Serotypes Isolated from the Cerebrospinal Fluid of Children with Suspected Meningitis: First Report From Alborz, Iran

Background: Pneumococcus causes various infections, some of which are life-threatening, including pneumonia, meningitis, otitis media, bacteremia, and sinusitis. Currently, more than 95 serotypes have been identified. Objectives: The purpose of this study was to isolate and determine Streptococcus pneumoniae serotypes from the cerebrospinal fluid (CSF) of children with suspected meningitis using the multiplex polymerase chain reaction (PCR) method. Materials and Methods: A total of 864 CSF samples were taken from children with suspected meningitis who were admitted to Imam Ali Hospital of Karaj for one year (January 2019 to January 2020). To collect positive S. pneumonia samples, the lytA gene was traced using specific primers and PCR techniques. Results: The results of this study indicated that only 16 patients have a pneumococcal infection (1.85%), of whom 50% have encapsulated pneumococci. Furthermore, serotypes 38 (12.5%), 7F (12.5%), 23F (25%), and 6A/B (50%) were the most common serotypes causing invasive pneumococcal infections in the included samples. Conclusion: Among the techniques for serotyping S. pneumoniae, the multiplex PCR technique is known as a fast, easy, and low-cost method that can serotype a large number of samples. The results also showed that the 13-valent pneumococcal conjugate vaccine can cover at least 50% of the strains that cause invasive pneumococcal diseases (IPDs), which are life-threatening, especially in children. Therefore, it is suggested that the healthcare administrators of Iran design and implement a public vaccination program.

Development of a real-time PCR and multiplex PCR assay for the detection and identification of mycotoxigenic fungi in stored maize grains

ABSTRACT This study aimed to identify important mycotoxigenic fungi and accurate detection of mycotoxin in stored maize grains using molecular methods. The current study also optimised the real-time PCR (RT-PCR) assay. The melting curve was established to identify isolated fungal species of Aspergillus (4), Fusarium (3), Penicillium (3), and Alternaria (one). A multiplex polymerase chain reaction (mPCR) technique was developed for the detection and characterisation of mycotoxin producing fungi, mycotoxin metabolic pathway genes, and the determination of eleven mycotoxins in stored maize grains using high-performance liquid chromatography (HPLC). The mPCR results indicated positive signals for potentially mycotoxigenic fungal species tested of Aspergillus, Fusarium, Penicillium, and Alternaria. A protocol for multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was tested to distinguish between free and contaminated, stored maize with aflatoxin B1 (AFB1). The expression pattern of four aflatoxin biosynthetic pathway genes, AFB1 (aflQ, aflP, aflO, and aflD), was a good marker for contaminated, stored maize grains. HPLC analysis showed that maize grain samples were contaminated with mycotoxins, and the concentration was above the detection level. The results indicate that the polyphasic approach might provide a sensitive, rapid, and accurate method for detecting and identifying mycotoxigenic fungal species and mycotoxins in stored maize grains.

Multiplex PCR for Detection of Exfoliative Toxins, and Toxic Shock Syndrome Toxin 1 Genes in MRSA Strains of Clinical Staphylococcus aureus Isolates

This research approaches to investigate toxin gene content of the methicillin resistant Staphylococcus aureus strains that are clinically collected from human skin infection cases by performing multiplex polymerase chain reaction (PCR) technique (irrespective of whether the strain produces the toxin or not) and for that a single PCR reaction was employed, two pairs of exfoliative toxin and one pair toxic shock syndrome toxin-1. Amplicon sizes ranged between 93, 226 and 326bp, to facilitate electrophoretic separation. Results of the current study had revealed that the major gene detected was tst found in six isolates. Multiple toxin gene combinations were also observed. This work was conducted at several institutions including Tikrit Teaching Hospital’s lab; college of science biology department labs/ Tikrit University; and the Biotechnology Research Centre/ Al-Nahrain University.

Prevalence of ovine theileriosis in Mosul city, Iraq

The present study aimed to determine the prevalence of ovine theileriosis (OT) in sheep in Mosul city, Iraq using microscopic examination (ME) of the blood smears stained with MGG- Quick stain and conventional polymerase chain reaction technique (c-PCR) to compare between c-PCR technique and ME as techniques for the diagnosis of disease, and to investigate the pattern and type of infections based on multiplex polymerase chain reaction technique (m-PCR). From October 2021 to May 2022, one-handed eighty-five Blood samples were drawn randomly from sheep in various regions of Mosul city. The overall prevalence of OT was 42% (22.7 out of 185) and 52.4% (97 out of 185) using microscopic examination and c-PCR technique, respectively. A slight agreement was observed between ME of blood smears and c-PCR technique according to Kappa value 0.190, with low sensitivity, specificity, and accuracy of ME method was 30%, 88.6%, 58.4%, respectively, compared with c-PCR technique. The prevalence of mixed infection 22.7% and single infection with T. lestoquardi 20% were significantly higher (P<0.05) than single infection with T. ovis 9.7%. This study concludes that OT is widespread in Mosul city, Iraq, and the c-PCR technique is more reliable and suitable for detecting Theileriainfection in sheep than the ME method.

Rapid solid phase microextraction of DNA using mesoporous metal-organic framework coating for PCR-based identification of meat adulteration.

A rapid, convenient, low-cost, and selective DNA isolation method was developed for identifying meat adulteration. A mesoporous metal organic framework (Meso-UIO-66)-coated solid phase microextraction system was employed as an isolation device to simplify DNA isolation into three steps (lysis, washing, and elution). Meso-UIO-66 was utilized as the adsorbent because of its positively charged surface, high chemical stability, and mesoporous structure. Meso-UIO-66 was characterized by scanning electron microscopy, transmission electron microscopy, powder X-ray diffraction, Fourier transform infrared spectroscopy, ultraviolet‒visible spectroscopy, X-ray photoelectron spectroscopy, and nitrogen adsorption-desorption tests. Parameters that affected DNA isolation were optimized. This method can be used to isolate and purify DNA from meat in 60s, and the DNA concentration and purity are comparable to those of samples isolated with a commercial kit. Multiple DNA detection was achieved by coupling this method with the multiplex polymerase chain reaction technique, and the detection limit was lower than 1% (w/w).

Panton-Valentine Leucocidin-Positive Staphylococcus aureus Nasal Carriers in a Tertiary Hospital in Selangor-Malaysia

Panton-Valentine Leukocidin (PVL) is a cytotoxin produced by Staphylococcus aureus that causes leukocyte destruction and tissue necrosis. Therefore, this study aimed to detect the rate, antimicrobial susceptibility, associated risk factors, and the phylogenetic relationship of PVL-positive S. aureus nasal carriers among patients and nurses. The research methods included the collection of 315 nasal specimens obtained from inpatients and nurses. The identification of S. aureus was confirmed by a coagulase test. The multiplex polymerase chain reaction technique was used to affirm PVL-positive S. aureus. The antibiotic sensitivity of S. aureus isolates was carried out using the disk diffusion method. The phylogenetic similarity of PVL-positive S. aureus was identified by pulsed-field gel electrophoresis. This study revealed that 160 out of 315 (50.8%) isolates were S. aureus. In addition, 7/160 (4.4%) had the lukS gene (six MSSA and one MRSA). The PVL-positive S. aureus isolates were 100% sensitive to gentamicin, linezolid, mupirocin, rifampin, trimethoprim-sulfamethoxazole, teicoplanin, tigecycline, and vancomycin. The risk factor analysis revealed that a longer hospital stay, nasogastric intubation, and runny nose were significant risk factors for patients to be PVL-positive S. aureus nasal carriers. A phylogenetic similarity analysis of PVL-positive isolates showed five models and they were distantly correlated. Therefore, the current study will provide knowledge to the hospital infectious control authority.

Detection and characterization of meat adulteration in various types of meat products by using a high-efficiency multiplex polymerase chain reaction technique.

Identification of meat authenticity is a matter of increasing concerns due to religious, economical, legal, and public health reasons. However, little is known about the inspection of eight meat species in one tube reaction due to technological challenge of multiplex polymerase chain reaction (PCR) techniques. Here, a developed multiplex PCR method can simultaneously authenticate eight meat species including ostrich (753 bp), cat (564 bp), goose (391 bp), duck (347 bp), chicken (268 bp), horse (227 bp), dog (190 bp), and sheep (131 bp). The detectable deoxyribonucleic acid (DNA) contents for each target species was as low as 0.01 ng in both raw and heat-treated meat or target meat down to 0.1% (w/w) of total meat weight reflecting high stability of the assay in heat processing condition, indicating that this method is adequate for tracing meat origin in real-world meat products, which has been further validated by authenticity assays of commercial meat products. Overall, this method is a powerful tool for accurate evaluation of meat origin with a good application foreground.

Design and Performance Test of Specific Primers to Detect Bovine DNA Fragments using Multiplex PCR Technique for Halal Authentification

Adulterating meat products with several species, including non-halal species, is often found in commercial products. Therefore, non-halal ingredients are a major source of concern for Muslims. Multiplex polymerase chain reaction (PCR) with multiple primers can detect contamination of meat components from non-halal species in a single reaction process, making it more effective and efficient. Multiplex PCR is a PCR technique that uses multiple primers and DNA samples in one reaction to amplify multiple target regions. In this study, a pair of species-specific primers encoding the Cytochrome c oxidase subunit I (CO1) gene were designed to amplify bovine DNA, tested for specificity, and applied in multiplex PCR technique together with D-loop primers for pigs, Cyt-b for rats, and 12S rRNA for dogs. The CO1 primers, along with D-loop primers for porcine, Cyt-b primers for rats, and 12S rRNA primers for dogs, can be used to detect specific bovine DNA with a size of 279 bp and sequence similarity of 96%.

The molecular identification of diarrheagenic Escherichia coli (DEC) isolated from meat and meat products

The present study aims to diagnose diarrheagenic E. coli in meat and meat products by the conventional polymerase chain reaction (PCR) technique using the uidA gene to confirm the existence of the bacterial isolates as E. coli. The multiplex PCR technique is adopted to detect the virulence genes of these bacteria using two groups of primers for detecting the gene (stx1, stx2, aggR, esth, eae, invE, daaC, estp, elt, and bfpA). This study applies these primers to 100 E. coli strains isolated from 782 samples of meat and meat products (fresh, minced, burger, pastirma, and chicken) from February to November 2020. The results of the present study show that all E. coli isolates are positive to have the uidA gene (147 bp). The study also detects 95/782 (12.15%) pathogenic species related to virulence genes by using multiplex PCR. The highest percentage of pathotype is ETEC, 46.32%, and the lowest is the DAEC type 1.05%. In addition, the other pathotypes are 20.05, 14.74, 6.32, 6.32, and 5.26% of STEC, EHEC, aEPEC, EAEC, and EIEC, respectively. The high contamination rate with DEC reported in this study is associated with the poor hygiene conditions of slaughtering and meat storage in shops and markets, resulting in health risks to consumers.

Development of a multiplex polymerase chain reaction technique for detection and discrimination of Eimeria spp. in cattle in Indonesia

Background and Aim:Bovine eimeriosis is a disease caused by apicomplexan parasites of the genus Eimeria. It is one of the most important and widespread bovine illnesses in the world. Some of the identified species of bovine eimeriosis have morphologically similar oocysts that are difficult to differentiate. For the identification of particular Eimeria spp., diagnostic laboratories are increasingly turning to DNA-based technology. This study aims to develop a multiplex polymerase chain reaction (mPCR) technique based on the internal transcribed spacer-1 (ITS-1) gene for the simultaneous identification of pathogenic Eimeria spp. in cattle from Sulawesi Island, Indonesia.Materials and Methods:Genomic DNA was extracted by the DNAzol reagent from the purified Eimeria oocysts. Species-specific primers targeting the ITS-1 region were used to amplify the distinct Eimeria spp.Results:Using PCR ITS-1, this study showed that 36 of 120 fecal samples (30%) were infected by Eimeria spp. The multiplex PCR assay allowed for the simultaneous identification of six major Eimeria spp. in a single-tube reaction. The proportion of mixed Eimeria spp. infections was 100% (36/36). The maximum number of Eimeria spp. was five, and the minimum number was two.Conclusion:Identification of six pathogenic Eimeria spp. in cattle was successfully carried out by nested multiplex PCR using ITS-1 gene. In the future, a procedure to detect pathogenic Eimeria spp. in one tube reaction will offer economical and save diagnostic time.

Comparing PCR With High-resolution Melting Analysis for Apolipoprotein E Genotyping in Alzheimer's: A Case-control Study.

The apolipoprotein E (APOE) genotype has a heterogeneous distribution throughout the world. The present study aimed to characterize the APOE genotype (rs429358, rs7412) in healthy individuals compared with Alzheimer cases in Kerman, southeastern Iran, by two standard mutation scanning methods. In this case-control study, 90 Alzheimer patients as a case group and 90 healthy individuals as a control group were examined. APOE genotyping was carried out using high-resolution melting (HRM) analysis assay and multiplex tetra-primer amplification-refractory mutation system polymerase chain reaction (T-ARMS PCR) techniques. In contrast to Multiplex T-ARMS PCR, HRM analysis was not efficient in rs7412 genotyping. The results of multiplex T-ARMS showed that ɛ2ɛ3 genotype (P=0.006, odd ratio [OR]=0.119) and ɛ2 allele (P=0.004, OR=0.129) were more prevalent in the control group compared with the case ones, whereas ɛ4 allele was associated with borderline risk of Alzheimer disease (P=0.099, OR=1.76). We concluded that Multiplex T-ARMS PCR could be considered as a better option than HRM analysis for APOE genotyping in terms of speed, accuracy, simplicity, and cheapness in large-scale use. Also, the present study revealed that ɛ2 ɛ3 genotype and ɛ2 allele are protective against Alzheimer whereas the ɛ4 allele cannot be strongly considered as Alzheimer genetic risk factor in Kerman, Iran. The results may help to choose a better technique for APOE genotyping.