Nested PCR Method Research Articles

Seasonal dynamics of haemosporidian (Apicomplexa, Haemosporida) parasites in house sparrows Passer domesticus at four European sites: comparison between lineages and the importance of screening methods

Infectious diseases often vary seasonally in a predictable manner, and seasonality may be responsible for geographical differences in prevalence. In temperate regions, vector-borne parasites such as malaria are expected to evolve lower virulence and a time-varying strategy to invest more in transmission when vectors are available. A previous model of seasonal variation of avian malaria described a double peak in prevalence of Plasmodium parasites in multiple hosts resulting from spring relapses and transmission to susceptible individuals in summer. However, this model was rejected by a study describing different patterns of seasonal variation of two Plasmodium spp. at the same site, with the double peak only apparent when these species were combined. Here, we assessed the seasonal variation in prevalence of haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) in house sparrows (Passer domesticus) sampled across 1 year at four temperate European sites spanning a latitudinal range of 17°. We showed that parasite prevalence and diversity decreased with increasing latitude, but the parasite communities differed between sites, with only one Plasmodium lineage (P_SGS1) occurring at all sites. Moreover, the nested PCR method commonly used to detect and identify haemosporidian parasites strongly underestimated co-infections of Haemoproteus and Plasmodium, significantly biasing the pattern of seasonal variation, so additional molecular methods were used. Finally, we showed that: (i) seasonal variation in prevalence of haemosporidian parasites varied between study sites and parasite lineages/species/genera, describing further cases where the double peak model is not met; (ii) the seasonal dynamics of single lineages (P_SGS1) varied between sites; and (iii) unexpectedly, seasonality was greatest at the southernmost site, a pattern that was mostly driven by lineage H_PADOM05. Limitations of the genotyping methods and consequences of pooling (parasite lineages, sites and years) in studies of haemosporidian parasites are discussed and recommendations proposed, since these actions may obscure the patterns of prevalence and limit ecological inferences.

The Burden of Toxoplasma Gondii In Spontaneous Miscarriage and its Association with Rhoptry Protein 5 Gene and Toxoplasma gondii specific primers GRA 6 Gene Expression in Iraq Women

Background: One of the major infectious micro-organism that is associated with spontaneous abortion is toxoplasmosis. Under diagnosis of toxoplasmosis and lack of understanding the exact mechanism and pathogenesis of spontaneous abortion associating toxoplasmosis are in our opinion the main problem facing clinicians who are concerned with categorization and management of spontaneous abortion in our pregnant ladies. Aim of the current study: to diagnose placental tissues parasitic infections using sophisticated nested PCR method in addition to evaluating gene expression of two pathogenetically Toxoplasma genes, namely ROP-5 and GRA-6, as a step toward estimating the exact prevalence of Toxoplasma associated spontaneous abortion in Iraqi community Patients and methods: The case control study was based on the inclusion of 101 women with spontaneous abortion and 20 pregnant ladies who succeeded to get pregnant serving as control group. The placental tissues were obtained from the pool of women visiting Al-Kut maternity teaching hospital for purpose of delivery or because of abortion. Those tissues were then processed in central laboratory using conventional PCR technique searching for evidence of toxoplasma gondii DNA. Obtained tissues were also subjected to real time PCR for detection of Rhoptry protein 5 gene expression and Toxoplasma gondii specific primers GRA 6. The lab work was done according to instruction of providing company. Results: Positive results were limited to women with spontaneous abortion, that is, none of control women had PCR evidence of Toxoplasma DNA in their placental tissues, 72 (71.3 %) versus 0 (0.0 %); the difference was highly significant (P < 0.001). An estimation of the risk of abortion accompanying toxoplasmosis has been carried out in terms of approximate Odds ratio and the results has been strikingly high (100.8). Both Rhoptry protein 5 and Toxoplasma gondii specific primers GRA 6 gene expressions have been significantly correlated to serum IgG and IgM positive tests in a negative way. Conclusion: PCR carried out on placental tissue is significantly higher sensitive in detecting toxoplasma gondii than serology and Both Rhoptry protein 5 and Toxoplasma gondii specific primers GRA 6 gene expressions play significant role in spontaneous abortion.

Diagnostic Performance of Three Rapid Diagnostic Test Kits for Malaria Parasite Plasmodium falciparum.

Malaria is a potent burden on public healthcare worldwide due to requiring rapid diagnosis and treatment. Nowadays, prompt diagnosis with rapid diagnostic tests (RDTs) has been widely accepted as an effective diagnostic technique in malaria-endemic countries, primarily due to their easy operation, fast output, and straightforward interpretation. The global availability and use of RDTs have gradually grown over recent decades as field-applicable diagnostic tests for the reliable confirmation of malaria infection and proper case management. This study was conducted to evaluate diagnostic performance of 3 commercially available malaria RDT kits : BIOCREDITTM Malaria Ag Pf(pLDH), Malaria Ag Pf(pLDH/pHRPII), and Malaria Ag Pf/Pv(pLDH/pLDH) (where pLDH and pHRPII stand for plasmodium lactate dehydrogenase and histidine-rich protein 2, respectively) for the specific detection of Plasmodium falciparum. A total of 1,129 blood samples including 95 blood samples, confirmed as vivax malaria infection by microscopic examinations and a nested-PCR method, were tested for falciparum malaria infection. The overall sensitivity and specificity of Malaria Ag Pf(pLDH/pHRPII), Malaria Ag Pf/Pv(pLDH/pLDH), and Pf(pLDH) for P. falciparum were 99.0% and 100%, 95.8% and 100%, and 100% and 100%, respectively. It is proposed that the 3 RDT kits perform reliable level of diagnostic accuracy of detection for P. falciparum parasites.

Prevalent Latent Human Cytomegalovirus Genotype b2 in Biopsy Samples of Gastric Cancer

Aim: Genotyping and prevalence of human cytomegalovirus (HCMV) in gastrointestinal tract remains unclear. Objective: Characterization of HCMV and its viral load in samples of patients with gastric cancer (GC) in northeast Iran. Methods: A total of 21 biopsies were collected from patients with GC. HCMV was detected and genotyped using multiplex nested PCR. Quantitative real-time PCR has been used to determine the viral load. Staining was performed to detect viral inclusions. Results: 76.19% of the samples had HCMV genotypic b2 with viral load <0.1 IU/μl. 66.67% of patients were positive for H. pylori. Conclusion: Multiplex nested-PCR method can detect CMV populations with low viral loads in GC biopsies. For the first time, it was determined that the genotype b2 of HCMV is prevalent in the gastrointestinal tract.

The Prevalence of Malaria in Soldiers, Zahedan, Iran

Background: Malaria is an infectious disease, which is caused by different species of plasmodium. It damages social and economic growth and is also one of the leading causes of morbidity and mortality. Malaria is transmitted by female anopheles mosquitoes. Studying the epidemiology of malaria is critical, and it has been poorly studied in asymptomatic individuals, especially in boundary soldiers. Objectives: This study aimed at evaluating the prevalence of malaria in veterans. Methods: In this cross-sectional study, a total of 300 blood samples were collected from the volunteers of a garrison in Zahedan City, Sistan and Baluchestan Province, Southeast of Iran during the spring and summer of 2017 and simultaneously, the questionnaires were completed by the subjects. Then, the samples were examined by microscopic examination and nested PCR method. Results: Of 300 studied samples, 4 samples (1.3%) were positive for P. vivax and had asymptomatic malaria, of which. Three samples were native to Sistan and Baluchistan and another one was native to Kerman. No species of Plasmodium was observed. Conclusions: A low prevalence (1.3%) of malaria indicated that it has decreased in Sistan and Baluchistan during the last years and also using the nested PCR technique is suggested to diagnose malaria in asymptomatic patients.

Comparison of Different 18S rRNA Primers with Conventional PCR Methods in Determination of Plasmodium spp. and Evaluation of Nested PCR Method

Comparison of Different 18S rRNA Primers with Conventional PCR Methods in Determination of Plasmodium spp. and Evaluation of Nested PCR Method

Comprehensive study on the molecular prevalence and seasonal and age distribution of Mycobacterium avium subspecies paratuberculosis in raw milk and traditional dairy products

Due to the high consumption rate of milk and dairy products, they should have a high microbial quality. Maycobacterium avium subspecies paratuberclosis is one of the emerging zoonotic bacteria with high economic importance. The present study was done to assess the prevalence of the MAP in different types of raw milk and traditional dairy samples. Seven-hundred and eighty-five raw milk and traditional dairy samples were collected. DNA was extracted using a DNA extraction kit. Detection of MAP was done using the IS900 nested-PCR method. Eighty-eight out of 785 (11.21%) raw milk and traditional dairy product samples harbored MAP. Prevalence of MAP in raw milk and traditional dairy samples were 12.38% and 8.84%, respectively. Raw buffalo (25%) milk samples had the highest prevalence of MAP, while raw donkey (6.66%) had the lowest. Traditional cheese (24%) samples had the highest prevalence of MAP, while traditional kashk (2.50%) had the lowest. Seasonal and age distributions were also observed for the prevalence of MAP. Raw milk and traditional dairy samples may be the sources of MAP transmission into the human population. Marked seasonality and age distribution were seen for the prevalence of MAP. Further studies are required to find more information about MAP infection in raw milk and traditional dairy products.

Nested PCR methods for detection Toxoplasma gondii B1 gene in Cerebrospinal Fluid of HIV patients

Background: Toxoplasmosis is a disease caused by infection of Toxoplasma gondii, Which may cause a life-threatening condition in immunocompromised patients, for example, Toxoplasma encephalitis (TE). It is challenging to diagnose Toxoplasma as a cause of central nervous system (CNS) infection in HIV patient, so we need an alternative method, which is a PCR detection of Toxoplasma gondii B1 gene.Objective: This research aimed to find association between PCR methods for Toxoplasma gondii B1 gene and anti-Toxoplasma IgG from cerebral spinal fluid patient HIV AIDS.Methods: A cross-sectional study was conducted to Cerebrospinal fluid (CSF) samples of HIV patients with neurological symptoms to determine Toxoplasma gondii infection using nested PCR methods for the B1 gene and detection of anti-Toxoplasma IgG.Results: 88 CSF samples from HIV patients tested using nested PCR showed 23 samples (26,1%) were positive. Serologic test for IgG Toxoplasma showed 34 samples were positive (28,6%). There was a significant correlation (p=0.000(<0.05) between PCR result and a serologic test for IgG Toxoplasma.Conclusion: Nested PCR methods to detect B1 gene increased the accuracy of diagnosis for toxoplasma encephalitis.

Adana İlinde Sığırlarda Anaplasma marginale ve Anaplasma centrale’nin Serolojik ve Moleküler Yöntemler ile Araştırılması

Anaplasmosis is a common disease in tropical and subtropical climate zone and is transmitted by vectors. Especially in large cattle management systems, it has started to be detected frequently in recent years. The aim of this study was to determine the prevalence of Anaplasma spp. in cattle in Adana province. For this aim, 187 blood samples were collected from cattle from fifteen districts of Adana that have different climatic zones and examined by Competitive ELISA (cELISA) and Nested-PCR methods. Seropositivity was determined as 38.5% (72/187) in cattle. The molecular prevalence was detected as 1.6% (3/187) for Anaplasma centrale and 3.2% (6/187) for Anaplasma marginale by Nested-Polymerase Chain Reaction (PCR) methods. In this study, epidemiological data related to bovine anaplasmosis in Adana province of Turkey were discussed in detail and it was thought that the obtained data would contribute to disease prevention and control programs.

Detection of Vibrio campbellii and V. parahaemolyticus carrying full-length pirABVp but only V. campbellii produces PirVp toxins

Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging disease of penaeid shrimps caused by a unique strain of Vibrio species that carries a plasmid harboring PirABVp-binary toxin gene. Since the first outbreak in 2009, the diseases have been reported in a number of countries in Asia, America, and Latin America. In the present study, we obtained 51 bacterial isolates recovered from five AHPND suspected shrimp farms in South America for PCR diagnosis using AP4 nested PCR method. There were 3/51 isolates (34, 36 and 43) from two farms which tested positive by AP4 primers targeting PirABVp toxin genes. The detection results were also confirmed by duplex pirABVp PCR assay. Subsequently, integrity of full length pirABVp genes in the three positive isolates was revealed by sequencing analysis. Bacterial species identification by ldh-specific PCR combined with multilocus sequencing analysis (MLSA) revealed that the two isolates 36 and 43 are V. parahaemolyticus while the isolate 34 is V. campbellii. Surprisingly, pirABVp mRNA transcript was detected from only V. campbellii 34 while that of V. parahaemolyticus 36 and 43 were undetectable. The results coincide with Western blot analysis that only V. campbellii 34 produces both PirAVp and PirBVp toxins while two isolates of V. parahaemolyticus pirAVp+ and pirBVp+ express neither PirAVp nor PirBVp toxins. Experimental challenge revealed that PirABVp-containing V. campbellii 34 and atypical V. parahaemolyticus isolates 36 and 43 were pathogenic to shrimp. Massive cell sloughing of hepatopancreatic tubule epithelial cells, characteristic of AHPND, was observed from shrimp exposed to isolate 34 while isolates 36 and 43 caused extensive collapsed hepatopancreatic tubule epithelia. The findings in this study indicated that there is a proportion of Vibrio isolates harboring intact pirABVp that were tested positive by PCR but did not produce AHPND PirABVp toxins. We thus suggested that investigation of pirABVp expression at transcriptional and translational levels as well as bioassay is required for confirmation of AHPND-causing Vibrio strain.

Detection of malaria with light microscopy and Nested polymerase chain reaction (Nested PCR) methods in peripheral blood expansions and investigation of the genetic diversity of Plasmodium species by 18S rRNA gene in Southeast of Iran

BackgroundMalaria is a public health concern that leads to about a million deaths worldwide every year. Malaria is caused by the genus Plasmodium, which includes P. falciparum, P. vivax, P. malariae, and P. ovale. Molecular phylogeny is essential to better recognition the evolution of the genus Plasmodium genus and detection of the relative degree of Plasmodium species in humans. The aim of this study was to detect malaria with Light Microscopy (LM) and Nested polymerase chain reaction (Nested PCR) methods in peripheral blood expansions and to investigate the genetic diversity of Plasmodium species by 18S rRNA gene in the southeast of Iran. MethodsA total of 97 blood smears were collected from patients suspected to malaria in a 6-year period in the southeast of Iran including Hormozgan, Kerman, and Sistan and Baluchestan provinces. Diagnosis of Plasmodium species on blood smears was performed using LM and Nested PCR methods. In addition, 16 Plasmodium-positive samples were chosen for the determination of genetic diversity. ResultsOverall, 97 of 97 (100%) studied cases were positive by LM while 94 of 97 (96.8%) of them were detected as malaria by Nested PCR. Except for seven cases, Nested PCR confirmed the LM results. These samples involved two P. vivax and five P. falciparum in the LM method. Meanwhile, the Nested PCR was detected in all of the cases as a mixed infection with P. vivax and P. falciparum. The results of the phylogenetic analysis revealed two main clades and five different subclades. About 87.5% of the isolates were located in clade I and contained P. vivax. In addition, 12.5% of the studied isolates involved P. falciparum that was in clade II. ConclusionAccording to our results, Nested PCR method had higher sensitivity than LM and is suggested as a good approach for malaria detection. Consideration the wide diversity of tested isolates and the importance of vaccine development, which is affected by this diversity, further studies are needed in this regard.

Toxoplasma gondii in Aborted Fetuses of Sheep in Ardebil Ar-ea, North-West of Iran

One of the severe complications of toxoplasmosis is the induction of abortion in sheep, goats, and human beings. The rate of abortions related to toxoplasmosis in sheep varies between 10% and 23% in the United States and European countries. In Iran, Toxoplasma infection were diagnosed in aborted ovine fetuses between 13.5% and 69% in the brain samples based on PCR. The purpose of this study was to investigate the prevalence of abortions related to T. gondii among the aborted fetuses of sheep in Ardabil area, North-West of Iran. The brains of 75 aborted sheep fetuses in Ardebil area were investigated with nested PCR method using fragments of the GRA6 gene during lambing seasons in 2014-2015. Meanwhile, thoracic and abdominal fluids of aborted fetuses and 200 serum samples collected from the herds affected by abortion were examined for the existence of anti-T. gondii IgG. For 48 out of the 75 fetal brain samples (64%), the results of nested PCR test were found to be positive. Furthermore, antibodies against Toxoplasma was observed in 136 (68%) collected samples of sheep and 21 samples (28%) of fetal fluids. There is the significant role of T. gondii in the abortions of sheep in Ardabil area. Meanwhile, this condition can also be dangerous for human beings because of their consumption of sheep meat.

Comparison of Nested-PCR and Loop-Mediated Isothermal Amplification for Cutaneous Leishmaniasis Diagnosis

Background: Leishmaniasis is a protozoan disease caused by Leishmania genus and its most common form is cutaneous leishmaniasis. The number of reported cases of cutaneous leishmaniasis in Khuzestan, southwest Iran, continues to increase. Therefore, early, accurate diagnosis is crucial for successful therapy. Objectives: The Nested PCR is a molecular method with high sensitivity and specificity in detecting leishmaniasis, but this method requires advanced equipment and skilled labor. Therefore, developing a simple yet accurate technique to diagnose leishmaniasis is essential. This study was designed to evaluate the possibility of replacing Nested PCR with loop-mediated isothermal amplification (LAMP) method for diagnosis of cutaneous leishmaniasis. Methods: We obtained 75 clinical samples from cutaneous leishmaniasis patients whose infection had already been confirmed by microscopic examination. The LAMP assay with pre-added malachite green was performed using a set of four primers targeting conserved sequences of the18S ribosomal RNA gene. The nested PCR method was performed using specific kinetoplast minicircle DNA primers. Cultured promastigotes of Leishmania major (MHOM /IR/75/ER), L. tropica (MHOM/IR/02/Mash10), and the virulent RH strain of Toxoplasma gondii were used as controls. Results: Our results showed that LAMP was positive for 100% of microscopically positive samples, which was similar to Nested PCR. The detection of L. major was improved at 104 parasites/ml using the LAMP method. Conclusions: Our findings suggest that considering the simplicity, specificity, and sensitivity of LAMP, it would be a potentially useful method in the diagnosis of leishmaniasis and detection of its various strains/species. This cost and time-effective method can be used as a suitable alternative for surveillance of leishmaniasis, as well as in epidemiological studies.

Association of HPV16 and 18 genomic copies with histological grades of cervical lesions.

The possible correlation between HPV16 and HPV18 genomic copies with the grade of cervical lesions needs more investigations. The aim of this study was to quantify genomic copies of HPV16 and 18 simultaneously and to find out the correlation between genomic copies numbers and different grades of lesions. Therefore, a total of 102 formalin-fixed and paraffin-embedded tissue specimens, 33 LSILI, 43 HSIL, and 26 squamous/adenocarcinoma were subjected to DNA extraction. The β-globin gene was selected to qualify the extracted DNA as well as normalization of viral titers using Taq-Man real-time PCR. The presence HPV16 and/or 18 were screened in tissue samples by nested PCR method, then an in- house Taq-Man Duplex real-time PCR assay was employed to quantify their genomic copies. The mean age of participants was 43 ± 13. Out of 102, 80 samples were positive for HPV16 and/or 18 DNA. There was a statistically significant association between HPV16 genomic copies and progression of cervical lesions (P < 0.001). In contrast, no such an association was found in the case of HPV18 (P = 0.51). Moreover, with 95% confidence intervals, 2.3-4 genomic copies of HPV16 genome/cell could be applicable to distinguish LSIL from HSIL and SCC. In conclusion, quantification of HPV16 genomic copy number showed a close association with progression of cervical lesion. Furthermore, HPV16 genomic copies of 4 copies/cell could be a set point to differentiate LSIL from HSIL.

Detection and Identification of Banana-associated Phytoplasma Using Nested-PCR Method

Phytoplasma is known to be associated with plant diseases in about 300 plant species from various families. Information on the presence of phytoplasma in bananas as one of the pathogens that can cause disease in bananas in Indonesia has never been reported. This research was conducted with the aim to detect the presence of banana phytoplasma by the nested-PCR method and to identify phytoplasma obtained based on the sequence analysis of the 16S rRNA gene. Standard PCR was carried out using P1/P7 primary pairs, followed by nested-PCR using a pair of R16F2n/R16R2m23SR primers separately that could amplify the target 16S rRNA genes in a row at 1600 bp. BLAST analysis shows that the results of phylogenetic analysis of banana phytoplasmic nucleotide cv. manggala from Tasikmalaya and cv. Raja nangka from Banjar has a genetic relationship that is closer to lethal wilt oil palm Phytoplasma (Candidatus Phytoplasma asteris). This phytoplasma belongs to the 16SrI-B group (aster yellows).

First Molecular Survey on Anaplasma phagocytophilum Re-vealed High Prevalence in Rural Dogs from Khuzestan Prov-ince, Iran

Anaplasmosis due to Anaplasma phagocytophilum is an important tick-borne zoonotic disease, which affects dogs, horses, cattle and human as well. This study aimed to probe the existence of this organism by means of molecular biology techniques for the first time in rural dogs of Khuzestan province, Southwestern Iran. During Sep 2014 to Apr 2015 blood samples of 103 apparently healthy rural dogs (60 males) were collected for A. phagocytophilum detection by light microscopical examination of Giemsa stained slides and Nested PCR on a fragment of 16S rRNA gene. From the examined slides, 11.65% were positive for A. morulae while 57.28% of infection was revealed by Nested PCR method. There was no statistical difference between ages and sexes of dogs and infection in molecular survey of A. phagocytophilum. Molecular prevalence of A. phagocytophilum was noticeably high. It may cause the incidence of disease in human population.

Genetic Clearness Novel Strategy of Group I Bacillus Species Isolated from Fermented Food and Beverages by Using Fibrinolytic Enzyme Gene Encoding a Serine-Like Enzyme.

Fibrinolytic enzyme gene (fibE) is widely conserved among Bacillus spp. belonging to group I species. This is encoding a serine-like enzyme (FibE) secreted in extracellular medium. This present work aims to assess the molecular usefulness of this novel conserved housekeeping gene among group I Bacillus spp. to identify and discriminate some related strains in traditional fermented food and beverages in Republic of Congo. First of all 155 isolates have been screened for enzymatic activities using caseinolytic assays. PCR techniques and nested PCR method using specific primers and correlated with 16S RNA sequencing were used. Blotting techniques have been performed for deep comparison with molecular methods. As a result B. amyloliquefaciens (1), B. licheniformis (1), B. subtilis (1), B. pumilus (3), B. altitudinis (2), B. atrophaeus (1), and B. safensis (3) have been specifically identified among 155 isolates found in fermented food and beverages. Genetic analysis and overexpression of glutathione S-transferases (GSTs) fused to mature protein of FibE in Escherichia coli BL21 and TOP10 showed 2-fold higher enzymatic activities by comparison with FibE wild type one. Immunodetection should be associated but this does not clearly discriminate Bacillus belonging to group I.

기문응애(Acarapis woodi) 특이 유전자 검출을 위한 초고속 nested PCR법 개발

기문응애(Acarapis woodi) 특이 유전자 검출을 위한 초고속 nested PCR법 개발

Gene identification and antimicrobial activity analysis of a novel lysozyme from razor clam Sinonovacula constricta

Lysozymes are important immune effectors present in phylogenetically diverse organisms. They play vital roles in bacterial elimination during early immune responses. In the present study, a second invertebrate-type (i-type) lysozyme gene from razor clam Sinonovacula constricta (denoted as ScLYZ-2) was cloned by RACE and nested PCR methods. The full-length cDNA sequences of ScLYZ-2 were 1558 bp, including a 5′ untranslated region (UTR) of 375 bp, an open reading frame of 426 bp, and a 3′-UTR of 757 bp with polyadenylation signal sequence (AATAAA) located upstream of the poly(A) tail. SMART analysis showed that ScLYZ-2 contains a signal peptide in the first 16 amino acid (AA) sequences and a destabilase domain located from 24 to 134 AA sequences. The deduced AA sequences of ScLYZ-2 were highly similar (42%–58%) to other known lysozyme genes of bivalve species. Multiple alignments of AA sequences showed that ScLYZ-2 possesses the classical i-type lysozyme family signature of two motifs [“MDVGSLSCGP(Y/F)QIK” and “CL(E/L/R/H)C(I/M)C”] and two catalytic residues (Glu35 and Asp46). Moreover, phylogenetic analysis showed that ScLYZ-2 is a new member of the i-type lysozyme family. In healthy razor clams, ScLYZ-2 was highly expressed in the hepatopancreas, followed by the gills, water pipes, and abdominal foot. Lysozyme activity and ScLYZ-2 expression levels were significantly upregulated in the hepatopancreas and gills after being infected with V. splendidus, V. harveyi, V. parahaemolyticus and S. aureus and M. luteus. Moreover, the recombinant ScLYZ-2 had strong antimicrobial activities against V. splendidus, V. harveyi, and V. parahaemolyticus. Furthermore, the minimal inhibitory concentration of the recombinant ScLYZ-2 against V. parahaemolyticus was 7.2 μmol/mL. Taken together, our results show that ScLYZ-2 plays an important role in the immune defense of razor clam by eliminating pathogenic microorganisms.

Analytical and clinical evaluation of a point-of-care molecular diagnostic system and its influenza A/B assay for rapid molecular detection of the influenza virus

Recently, rapid molecular detection systems have been used for point-of-care testing for the diagnosis of influenza worldwide. Here, we evaluated the performance of the cobas Liat system and the cobas Influenza A/B assay (Liat) using fresh nasopharyngeal samples collected from a Japanese population between December 2017 and February 2018. The performance of the examination was compared with that of antigen testing and a conventional polymerase chain reaction (nested-PCR) method. A total of 159 patients were included in this study, and 77 tested positive using Liat. The concordance rate between Liat and nested PCR was 97.5%. The median time between the ordering of testing and completion of molecular analyses using Liat was 30 min (interquartile range: 28–35 min). The overall sensitivity and specificity of antigen testing were 57.1% and 100%, respectively. The duration from symptom onset to examination did not alter antigen testing sensitivity. The current study demonstrates the high performance of Liat for the rapid molecular identification of the influenza virus.