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Internal reference genes are a prerequisite for ensuring the accuracy of gene verification experiments, but few relevant studies on Lyophyllum decastes have investigated the growth cycle and different environmental conditions. In this study, the qPCR results of 22 house-keeping genes were analyzed using GeNorm, BestKeeper, NormFinder and RefFinder. The results revealed that the most stable gene differed under different conditions. Across all developmental stages and under hot, cold, acidic, alkaline, and salt conditions, UBCE gene displays the greatest expression stability. However, EF1b, β-ACT, HSD17B3, and Cyb presented the greatest stability under cold, heat, and acidic conditions, and heavy metal exposure, respectively. To screen for genes suitable for all conditions, RefFinder’s ranking results revealed that UBCE and EF1b ranked in the top 2, demonstrating the highest gene expression stability. In contrast, Cyb was positioned at the bottom of the comprehensive ranking table. This study not only revealed potential factors affecting the suitability of reference genes but also identified optimal reference genes from a set of candidate genes across diverse conditions.

The color of strawberry fruit is an important appearance quality index that affects the marketability of fruit, and the content and type of anthocyanin are two of the main reasons for the formation of fruit color. At present, the research on anthocyanin synthesis mainly focuses on the phenylpropane metabolic pathway, and the F3H gene family is an important member of this metabolic pathway. Therefore, in order to clarify the role of flavanone 3-hydroxylase (F3H) in regulating anthocyanin accumulation in strawberry, we identified F3H gene family members in strawberry and analyzed their bioinformatics and expression at different fruit color stages. The results showed that the strawberry F3H family contains 126 members, which are distributed on seven chromosomes and can be divided into six subgroups. The promoter region of strawberry F3H gene family contains light response elements, abiotic stress response elements and hormone response elements. Intraspecic collinearity analysis showed that there were six pairs of collinearity of the F3H gene. Interspecific collinearity analysis showed that there were more collinearity relationships between strawberry and apple, grape and Arabidopsis, but less collinearity between strawberry and rice. Via tissue-specific expression analysis, we found that the expression levels of FvF3H48, FvF3H120 and FvF3H74 were higher in the stages of germination, growth, flowering and fruit setting. The expression levels of FvF3H42 and FvF3H16 were higher in seeds. The expression levels of FvF3H16 and FvF3H11 were higher in the ovary wall of stage 1, stage 2, stage 3 and stage 5. FvF3H15 and FvF3H48 were highly expressed in the pericardium, anther, receptacle and anther. Real-time fluorescence quantitative PCR showed the expression changes in F3H in the fruit coloring process. The results indicate that the expression levels of most members were higher during the S3 stage, such as FvF3H7, FvF3H16, FvF3H32, FvF3H82, FvF3H89, FvF3H92 and FvF3H112. FvF3H63 and FvF3H104 exhibited particularly high expression levels during the S1 stage, with some genes also showing elevated expression during the S4 stage, including FvF3H13, FvF3H27, FvF3H66 and FvF3H103. FvF3H58, FvF3H69, FvF3H79 and FvF3H80 showed higher expression levels during the S2 stage. These findings lay the groundwork for elucidating the biological functions of the strawberry F3H gene family and the selection of related genes.

The CorA / MGT / MRS2 family proteins are an important group of magnesium transporter proteins that maintain magnesium ion homeostasis in plant cells. However, little is known about the MGT functions in wheat. The known MGT sequences were used as queries to BlastP against wheat genome IWGSC RefSeq v2.1 assembly (E-value <10-5). Chromosome localization information for each gene was obtained from the GFF3 file of the wheat genome data (IWGSCv2.1).The sequence of 1500 bp upstream of the genes was extracted from the wheat genome data. The cis-elements were analyzed using PlantCARE online tool. A total of 24 genes were identified on 18 chromosomes of wheat. After functional domain analysis, only , , and had GMN mutations to AMN, while all the other genes had conserved GMN tripeptide motifs. Expression profiling showed that the genes were differentially expressed under different stresses and at different growth and development stages. The expression levels of and were significantly up-regulated in cold damage. In addition, qRT-PCR results also confirmed that these genes are involved in the wheat abiotic stress responses. In conclusion, The results of our research provide a theoretical basis for further research on the function of gene family in wheat.

Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 °C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = −3.2751x + 31.269 with a correlation coefficient R2 = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming.

Objective To analyze the clinical diagnostic value for thalassemia genotyping using peripheral blood mRNA. Methods Total RNA was extracted from 103 pre‐genotyped peripheral blood samples, followed by cDNA synthesis. These samples comprised 21 wild‐type cases, 49 α‐globin gene mutant cases and 33 HBB mutant cases. An Integrated strategy was adopted, combining single‐reaction Sanger sequencing with the multiple quantitative fluorescence PCR (MQF‐PCR), to analyze the full‐length coding sequences and expression level of HBA2/HBA1/HBB respectively. Results (1) The substitution mutations located in the coding region (n = 14) were successfully detected, and the mutations located in the HBB non‐coding region displayed negative results (n = 13). The samples with β41–42M/βN (n = 10) and β71–72M/βN (n = 4) were not observed to have their small deletion or insertion mutation initially, but they were eventually identified after using allele‐specific MQF‐PCR. Moreover, MQF‐PCR results indicated that the level ranges of Peak area ratio 1 (PAR1, PAHBA2/PAHBA1) and Peak area ratio 2 (PAR2, PAHBB/PAHBA) can be used to identify the common α‐globin gross‐deletion types and to predict mutations in the HBB non‐coding region. Conclusion This mRNA‐based strategy enabled detection of coding‐region mutations in the HBA2/HBA1/HBB and prediction of non‐coding‐region mutations as well as gross deletions with high coverage, low cost and simple operation. We have developed an integrated mRNA‐based strategy for thalassemia genotyping. Its characteristics included using only three Sanger sequencing reactions to analyze the full‐length coding sequence of HBA2, HBA1 and HBB, and employing a new multiple quantitative fluorescence PCR to simply assess and present the impact of common mutations on the expression levels of the three genes.

The bovine respiratory disease complex poses a significant threat to the cattle industry, necessitating a multifaceted approach to address its occurrence. The syndrome is caused by various pathogens such as bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3), bovine viral diarrhea virus (BVDV), bovine adenovirus type 3 (BAV3), (Mb), and infectious bovine rhinotracheitis virus (IBRV). The confluence of these pathogens causes substantial economic losses to the cattle industry. Although preventive and control measures have been implemented, containment of bovine respiratory diseases continues to present a formidable challenge, highlighting the need for innovative diagnostic and intervention strategies. In this study, we designed specific primers targeting six conserved pathogen genes ( of BRSV, of BPIV3, of BVDV, of BAV3, of Mb, and of IBRV). Subsequently, we established a multiplexed fluorescent real-time quantitative PCR (qPCR) assay for simultaneous detection of these pathogens. The developed method exhibited high specificity and sensitivity, with the lowest detection limits for plasmid DNA standards of BRSV, BPIV3, BVDV, BAV3, Mb, and IBRV being 70.1, 40.4, 15.1, 74.4, 69.6, and 4.99 copies/μL, respectively. The coefficients of variation determined by the assay established in this study were <4%, and the amplification efficiency was 93.84%-111.60%, which showed the reliability and stability of the method. The detection rates for BRSV, BPIV3, BVDV, BAV3, Mb, and IBRV were 7.59% (17/224), 11.61% (26/224), 8.04% (18/224), 22.32% (50/224), 27.23% (61/224), and 8.04% (18/224), respectively. All 224 cows were cases of natural disease. Fifty-six diseased cattle were infected with a mixture of two or more of the six pathogens at a mixed infection rate of 25% (56/224). Therefore, this study successfully developed a highly efficient, rapid, specific, and sensitive multiplex qPCR method to detect major pathogens associated with bovine respiratory diseases. This advancement is expected to significantly influence the future of the cattle industry and serve as a valuable reference for subsequent research in this field.

(SS) is one of the most important pathogens causing major economic losses in the global pig farming industry and is a serious threat to public health safety. It has multiple serotypes, with poor cross-protection between serotypes, and effective typing methods are lacking. In this study, a quadruplex TaqMan fluorescence quantitative PCR assay that can differentiate between types 2, 7 and 9 was developed using the gene, a generic gene for , and , and , genes encoding podocarp-associated genes for types 2, 7 and 9, respectively, as targets. The method is specific enough to accurately type pigmentosus without detecting non-target pathogens ( , , , and et al). The sensitivity was high, with a minimum lower detection line of 10 copies for P-SS and P-SS9, and 100 copies for P-SS2 and P-SS7. The standard curves generated showed good linearity with R of 0.999, 0.999, 0.997 and 0.998 respectively. The repeatability was good, with coefficients of variation between batch to batch and batch to batch tests ranging from 0.21% to 1.10%. Testing of 156 samples yielded 68 positive and 88 negative samples, of which the positive rate of SS was 5.77% (9/156), SS2 was 20.51% (32/156), SS7 was 8.33% (13/156) and SS9 was 9.6% (15/156), which was in line with the existing fluorescent quantitative PCR assay of 93.75%~100%, which was higher than the detection rate of conventional PCR. The quadruplex TaqMan fluorescence quantitative PCR method of generic, type 2, 7 and 9 established in this study can accurately differentiate the three serotypes of that currently have high prevalence and pathogenicity, which is of great importance for accurate clinical prevention and treatment, epidemiological investigation and vaccine development.

Soil contamination with heavy metals is a worldwide problem especially in China. The interrelation of soil bacterial community structure, antibiotic resistance genes, and heavy metal contamination in soil is still unclear. Here, seven agricultural areas (G1–G7) with heavy metal contamination were sampled with different distances (741 to 2556 m) to the factory. Denaturing gradient gel electrophoresis (DGGE) and Shannon index were used to analyze bacterial community diversity. Real-time fluorescence quantitative PCR was used to detect the relative abundance of ARGs sul1 , sul2 , tetA , tetM , tetW , one mobile genetic elements (MGE) inti1 . Results showed that all samples were polluted by Cadmium (Cd), and some of them were polluted by lead (Pb), mercury (Hg), arsenic (As), copper (Cu), and zinc (Zn). DGGE showed that the most abundant bacterial species were found in G7 with the lightest heavy metal contamination. The results of the principal component analysis and clustering analysis both showed that G7 could not be classified with other samples. The relative abundance of sul1 was correlated with Cu, Zn concentration. Gene sul2 are positively related with total phosphorus, and tetM was associated with organic matter. Total gene abundances and relative abundance of inti1 both correlated with organic matter. Redundancy analysis showed that Zn and sul2 were significantly related with bacterial community structure. Together, our results indicate a complex linkage between soil heavy metal concentration, bacterial community composition, and some global disseminated ARG abundance.

Haemonchus contortus poses a severe hazard to the healthy development of the sheep industry and threatens the welfare of sheep. Ivermectin is the primary anthelmintic used for the prevention and treatment of H. contortus parasitism. However, the widespread and uncontrolled application of ivermectin has resulted in the development and spread of resistant strains of H. contortus. P-glycoprotein (P-gp) plays important roles in the pharmacology and toxicology of ivermectin, and changes in P-gp expression levels can be used to analyze the resistance of H. contortus to ivermectin. This study aimed to analyze the effects of ivermectin on P-gp expression in H. contortus L3 larvae isolated from China and to evaluate whether changes in P-gp expression levels can be used to analyze resistant H. contortus strains. In the absence of drug treatment, the ivermectin-resistant strains isolated in China showed increased expression of P-gp11 (p < 0.01) compared with sensitive strains from elsewhere, whereas the expressions of P-gp2 and P-gp9.1 were downregulated (p < 0.01). When the same strain was compared before and after drug treatment, obvious differences in expression were observed between the different strains. Ivermectin-induced P-gp expression was found to be very complex among the L3 larvae of different strains. In addition, it was confirmed that using P-gp to determine ivermectin resistance in H. contortus strains from different geographic environments can yield different results.

Objective MiR-139-5p is a common tumor-associated microRNA (miRNA), which inhibits the occurrence and development of malignant tumors from various tissue sources. We detected miR-139-5p expression levels in tissues from patients with non-small cell lung cancer (NSCLC) to explore the relationship between miR-139-5p expression and clinicopathological parameters. Methods MiR-139-5p expression levels were detected in cancerous and normal tissues from 60 NSCLC patients by fluorescence quantitative polymerase chain reaction, using normal paracancerous tissue as a control. The relationships between miR-139-5p and clinicopathological parameters of NSCLC, including survival, were analyzed by t-tests and univariate analysis. Results MiR-139-5p expression levels were significantly reduced in NSCLC tissues compared with normal adjacent tissue. MiR-139-5p expression was not significantly associated with age, sex, or smoking history, but was related to clinical stage, pathological type, tumor size, and lymph node metastasis. Furthermore, low expression of miR-139-5p, clinical stage (II/III), adenocarcinoma, tumor ≥3 cm, and lymph node metastasis were all related to overall survival. Conclusion MiR-139-5p expression levels are down-regulated in NSCLC tissues, and low expression is associated with clinical stage, pathological type, tumor size, and lymph node metastasis in NSCLC patients. MiR-139-5p may act as a tumor suppressor gene in the occurrence and development of NSCLC.