Formic acid enhances whole-plant mulberry silage fermentation by boosting lactic acid production and inhibiting harmful bacteria.

Mulberry has also been regarded as a valuable source of forage for ruminants. This study was developed to investigate the impact of four additives and combinations thereof on fermentation quality and bacterial communities associated with whole-plant mulberry silage. Control fresh material (FM) was left untreated, while other groups were treated with glucose (G, 20 g/kg FM), a mixture of Lactobacillus plantarum and L. buchneri (L, 106 CFU/g FM), formic acid (A, 5 mL/kg FM), salts including sodium benzoate and potassium sorbate (S, 1.5 g/kg FM), a combination of G and L (GL), a combination of G and A (GA), or a combination of G and S (GS), followed by ensiling for 90 days. Dry matter content in the A, S, GA, and GS groups was elevated relative to the other groups (p < 0.01). Relative to the C group, all additives and combinations thereof were associated with reductions in pH and NH3-N content (p < 0.01). The A groups exhibited the lowest pH and NH3-N content at 4.23 and 3.27 g/kg DM, respectively (p < 0.01), whereas the C groups demonstrated the highest values at 4.43 and 4.44 g/kg DM, respectively (p < 0.01). The highest levels of lactic acid were observed in the GA and A groups (70.99 and 69.14 g/kg DM, respectively; p < 0.01), followed by the GL, L, and GS groups (66.88, 64.17 and 63.68 g/kg DM, respectively), with all of these values being higher than those for the C group (53.27 g/kg DM; p < 0.01). Lactobacillus were the predominant bacteria associated with each of these samples, but the overall composition of the bacterial community was significantly impacted by different additives. For example, Lactobacillus levels were higher in the G, A, and GA groups (p < 0.01), while those of Weissella levels were raised in the L, GL, and GS groups (p < 0.01), Pediococcus levels were higher in the A and GA groups (p < 0.01), Enterococcus levels were higher in the G and S groups (p < 0.01), and Lactococcus levels were raised in the S group (p < 0.01). Relative to the C group, a reduction in the levels of undesirable Enterobacter was evident in all groups treated with additives (p < 0.01), with the greatest reductions being evident in the A, S, GA, and GS groups. The additives utilized in this study can thus improve the quality of whole-plant mulberry silage to varying extents through the modification of the associated bacterial community, with A and GA addition achieving the most efficient reductions in pH together with increases in lactic acid content and the suppression of undesirable bacterial growth.

Comparative Analysis of Intestinal Inflammation and Microbiota Dysbiosis of LPS-Challenged Piglets between Different Breeds.

Post-weaning diarrhea is common in piglets, causing huge economic losses worldwide. Associations between LPS challenge, intestinal inflammation, and microbiota have been reported in Duroc × Landrace × Yorkshire (DLY) crossbred pigs. However, the effects of LPS challenge in other breeds remain unclear. In the current study, we performed a comprehensive comparative analysis of the effects of LPS challenge on jejunal mucosal morphology, jejunal microbial composition, and serum indexes in two pig breeds: DLY and Heigai, an indigenous Chinese breed. The results showed that LPS caused considerable damage to the mucosal morphology, enhanced serum levels of inflammatory cytokines and the intestinal permeability index, and lowered the antioxidant capacity index. LPS challenge also changed the microbial composition and structure of the jejunum, significantly increased the abundances of Escherichia-Shigella in DLY pigs, and decreased those of Gemella and Saccharimonadales in Heigai pigs. Furthermore, LPS challenge triggered functional changes in energy metabolism and activities related to the stress response in the jejunal bacterial community, alleviating the inflammatory response in Heigai pigs. This study also revealed that Heigai pigs had a weaker immune response to LPS challenge than DLY pigs, and identified several genera related to the breed-specific phenotypes of Heigai pigs, including Gemella, Saccharimonadales, Clostridia_UCG_014, Terrisporobacter, and Dielma. Our collective findings uncovered differences between Heigai and DLY pigs in intestinal inflammation and microbiota dysbiosis induced by LPS challenge, providing a theoretical basis for unraveling the mechanism of intestinal inflammation in swine and proposing microbial candidates involved in the resistance to diarrhea in piglets.

Dietary of different forms of Humulus scandens on growth performance and intestinal bacterial communities in piglets.

The present study was aimed at elucidating the effects of feeding different forms of Humulus scandens (Hu) on performance and bacterial communities in piglets. A total of 160 piglets were divided into four groups: (1) a control (CG, basal diet); (2) a basal diet with Hu pulp (HS), basal diet + Hu pulp; (3) a basal diet with Hu juice (HSJ), basal diet + Hu juice; and (4) a basal diet with Hu residue (HSR), basal diet + Hu residue. Results showed that HS, HSJ, and HSR supplementation led to rich average daily gain (ADG) and poor feed conversion ratio (FCR) during 28 to 70 d of age, increased 120 d body weight (BW), average daily feed intake (ADFI) and ADG and decreased FCR during 71 to 120 d of age. Three experiment groups presented greater (P < 0.05) IgA, IgG, and IgM and lower (P < 0.05) glucose, and blood urea nitrogen. The content of diamine oxidase significantly decreased (P < 0.05) in HS group. The crude  protein and crude fiber digestibility were improved (P < 0.05) in HS group and the Ca digestibility was increased (P < 0.05) in HS and HSJ groups. HSR supplementation improved the abundance of Firmicutes and decreased the abundance of Bacteroidetes. Hu supplementation with different forms increased the proportion of Lactobacillus in cecum content. These results indicated that supplemental feeding of Hu with different forms improved serum immunity, nutrient digestibility, and bacterial communities in piglets, promoting growth and development, which may be regarded as a reference for developing novel feed resources for piglets.

Comparative Analysis of Structural Composition and Function of Intestinal Microbiota between Chinese Indigenous Laiwu Pigs and Commercial DLY Pigs.

Intestinal microbiota has an important impact on pig phenotypes. Previous studies mainly focused on the microbiota of feces and worldwide farmed commercial pigs, while research on the microbiota of various intestinal sections and indigenous pig breeds is very limited. This study aimed to characterize and compare the biogeography of intestinal microbiota in pigs of one Chinese indigenous breed and one commercial crossbred. In this study, we sequenced the microbiota of six intestinal segments in the grown-up pigs of a Chinese indigenous breed, Laiwu pigs, and the worldwide farmed crossbred Duroc × Landrace × Yorkshire (DLY) pigs by 16S rRNA sequencing, characterized the biogeography of intestinal microbiota, and compared the compositional and functional differences between the two breeds. The results showed that there were obvious differences in microbial structure and abundance between the small and large intestines. Laiwu pigs had higher large intestinal diversity than DLY pigs, while DLY pigs had higher small intestinal diversity than Laiwu pigs. Moreover, some specific bacterial taxa and Kyoto Encyclopedia of Genes and Genomes pathways were found to be related to the high fat deposition and good meat quality of Laiwu pigs and the high growth speed and lean meat rate of DLY pigs. This study provides an insight into the shifts in taxonomic composition, microbial diversity, and functional profile of intestinal microbiota in six intestinal segments of Laiwu and DLY pigs, which would be essential for exploring the potential influence of the host's genetic background on variation in microbiota composition and diversity.

Effects of supplementing with Humulus scandens on the growth performance and gut microbiota in piglets.

AIMS: This study was conducted to evaluate the effects of feeding Humulus scandens (Hu) on growth performance and gut microbiota in piglets. METHODS AND RESULTS: A total of 120 piglets were allocated to four dietary treatments (1) CON, basal diet; (2) T1, basal diet + 2.0% Hu; (3) T2, basal diet + 2.8% Hu and (4) T3, basal diet + 3.6% Hu. The results showed that dietary H. scandens supplementation increased the final body weight and average daily gain. Furthermore, H. scandens supplementation in T1 groups increased the content of total protein, globulin and IgG in serum and the apparent digestibility of crude protein. Gut microbiota analysis showed that H. scandens treatment in T1 groups increased the abundances of Lactobacillus, Ruminococcaceae, Enterococcus and Pseudomonas in cecum content. CONCLUSIONS: These findings suggested that dietary H. scandens supplementation improved the growth performance, immunological function and nutrient apparent digestibility as well as modulating the gut microbiota in piglets. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributed to developing new feed resources and might provide an alternative strategy for growth promotion in piglets.

Spatial heterogeneity of particulate organic matter for the sorption of ciprofloxacin at the microstructure scale.

Particulate organic matter (POM) is a major antibiotic sorbent of the active soil organic carbon pool. Until now, the spatial heterogeneity of POM for ciprofloxacin (CFC) sorption at the microstructure scale has not been quantified. To our knowledge, this is the first study that combines a batch sorption experiment, NanoSIMS, and a 13C isotopic tracer to expound the distribution characteristics of CFC and the dominant sorption components of POM. Four POMs separated from soils (wetland, oil waste field, and farmlands) were utilized to study the sorption mechanisms using batch experiments. POM separated from the wetland was utilized for further study at the microstructure scale. The results revealed that the POM had a great CFC sorption capacity ranging from 65.20 mg g-1 to 77.51 mg g-1. Both the kinetics and NanoSIMS results showed that the sorption mechanisms included surface distribution and intra-particle diffusion. The microstructural distribution characteristics exhibited a marginal accumulation tendency and a surface accumulation effect. A combination of the NanoSIMS with the pH and salinity results explained that CFC adsorption was dominated by the organic components of POM. Overall, the present study demonstrates that the characteristics and organic components of the POM played a decisive role in the CFC-POM interactions. This study provides the first evidence of the vector potential of CFC rereleased into the environment during the POM turnover process.

Dynamic transcriptome profiles of postnatal porcine skeletal muscle growth and development.

BACKGROUND: Skeletal muscle growth and development are closely associated with the quantity and quality of pork production. We performed a transcriptomic analysis of 12 Longissimus dorsi muscle samples from Tibetan piglets at four postnatal stages of 0, 14, 30, and 60 days using RNA sequencing. RESULTS: According to the pairwise comparisons between the libraries of the muscle samples at the four postnatal stages, a total of 4115 differentially expressed genes (DEGs) were identified in terms of |log2(fold change)| ≥ 1 and an adjusted P value < 0.01. Short-time series expression miner (STEM) analysis of the DEGs identified eight significantly different expression profiles, which were divided into two clusters based on the expression pattern. DEGs in cluster I displayed a pattern of decreasing to a nadir, and then a rise, and the significantly enriched gene ontology (GO) terms detected using them were involved in multiple processes, of which the cell cycle, immunocyte activation and proliferation, as well as actin cytoskeleton organization, were the top three overrepresented processes based on the GO terms functional classification. DEGs in cluster II displayed a pattern of increasing to a peak, then declining, which mainly contributed to protein metabolism. Furthermore, besides the pathways related to immune system, a few diseases, and protein metabolism, the DEGs in clusters I and II were significantly enriched in pathways related to muscle growth and development, such as the Rap1, PI3K-Akt, AMPK, and mTOR signaling pathways. CONCLUSIONS: This study revealed GO terms and pathways that could affect the postnatal muscle growth and development in piglets. In addition, this study provides crucial information concerning the molecular mechanisms of muscle growth and development as well as an overview of the piglet transcriptome dynamics throughout the postnatal period in terms of growth and development.

[Isolation, identification and heavy metals biosorption of a lead and cadmium-tolerant strain].

Adding biological passivation agent during composting is one of the most effective ways to reduce the toxicity of heavy metals in contaminated livestock manure. To further improve biological passivation, we obtained a strain with high-heavy metal compounds tolerance to passivate heavy-metal contaminated manure and to characterize heavy-metal biosorption. High-tolerance microorganisms for lead and cadmium were isolated and screened from swine manure composting samples. The strain was identified by its morphology and molecular biology. After the influence of different pH, temperature and salt concentrations on growth of the strain were investigated, the optimal growth conditions were obtained for further analysis of its biosorption characteristics of lead and cadmium. The bacterium with tolerance to lead and cadmium termed SC19 was obtained, whose lead resistance was 600 mg/L and cadmium resistance was 120 mg/L. The isolate was further identified as Cedecea sp., and then its optimum pH was 7.0, temperature was 37 °C, and salt concentration was 0.5%. Lead removal was highest after 30 min of adsorption by the SC19 strain cultured for the stationary phase 36 h, and the maximum removal rate and biosorption capacity of lead were 60.7% and 329.13 mg/g, respectively. Meanwhile, cadmium removal was highest after 30 min of adsorption by the strain cultured for the logarithmic phase 8 h, and the maximum removal rate and biosorption capacity of cadmium were 51.0% and 126.19 mg/g, respectively. Fourier Transform InfraRed (FT-IR) results revealed that the biosorption process mainly happened on the surface of SC19 cell and many active groups on the cell surface could chelate the Pb²âº and Cd²âº. By comprehensive comparison, it was showed that strain SC19 shared a certain capacity of Pb²âº and Cd²âº biosorption, and the bacterium provided precious microbial germplasm resources for biological passivation of heavy metal contaminated manure.

[Effects of microbial agents on bacterial community composition during swine manure composting]. / å¾®ç”Ÿç‰©èŒå‰‚å¯¹çŒªç²ªå †è‚¥ä¸­ç»†èŒç¾¤è½ç»“æž„çš„å½±å“.

The process of swine manure and wheat straw aerobic composting was examined, with exogenous microbial agents being added in treatment group. The physicochemical properties were measured by conventional methods, and bacterial community characteristics were investigated by high throughput sequencing analysis. Exogenous microbial agents increased high-temperature duration, reduced pH value at the end of fermentation stage, augmented total nitrogen content, reduced C/N ratio. Results from principal component analysis showed that microbial agents affected the stability of bacterial community during composting. At the phylum level, the relative abundance of Firmicutes, Proteobacteria, and Chloroflexi was higher in the treatment group. At the class level, the relative abundance of Clostridia, Alphaproteobacteria, and Gammaproteobacteria in the treatment group were higher at the mesophilic and thermophilic phases. At the family level, Peptostreptococcaceae, Clostridiaceae_1, and Halanaerobiaceae of the Clostridia and Micromonosporaceae in the treatment group were higher at the mesophilic and thermophilic phases. Halocella was significantly positively correlated with exogenous microbial agents, while Ammoniibacillus was significantly negatively correlated with it. It suggested that microbial agents significantly changed the physicochemical properties and bacterial community structure during swine composting.

Sorption of oxytetracycline in particulate organic matter in soils and sediments: Roles of pH, ionic strength and temperature.

Particulate organic matter (POM) is a fraction of organic matter with dissimilar properties in different soils. POM isolated from soils and sediments (wetland, oil waste field, farmlands and aquaculture pond sediment) was used to study its sorption behavior on the antibiotic oxytetracycline (OTC). Impacts of solution pH, ionic strength and temperature on the OTC sorption were studied. The sorption rates of OTC in POM from wetland (POM-w) and farmland (POM-f1, POM-f2) were rapid during the first 3 h and gradually decreased with reaction time until reaching the equilibrium. Linear sorption occurred from 3 to 12 h in POM from oil waste field land (POM-o) and aquaculture pond sediment (POM-a). The organic carbon normalized partition coefficient (koc) varied from 215.0 to 4493.6 L kg-1, and it was nearly 10× higher for the POM-w, POM-f1 and POM-f2 than in the POM-o and POM-a. Sorption of OTC by POM exhibited strong pH dependence. Ionic factors affected OTC sorption in POM-f1, POM-f2 and POM-a. The sorption capacity declined >50% in a solution with Ca2+ compared to other ions with similar ionic strength. Sorption thermodynamics showed an entropy increasing and endothermic progress during the OTC sorption in POM, implying a spontaneous sorption process. Several mechanisms were involved in OTC sorption in POM, including hydrogen bonding, cation exchange, hydrophobic partitioning and surface complexation.

Comparative gene expression profiling of muscle reveals potential candidate genes affecting drip loss in pork.

BACKGROUND: Drip loss is a key aspect of meat quality. Transcriptome profiles of muscle with divergent drip loss would offer important insight into the genetic factors responsible for the trait. In this study, drip loss and other meat quality traits of 28 purebred Duroc pigs were measured, muscles of these individuals were RNA sequenced, and eight individuals with extremely low and high drip loss were selected for analyzing their transcriptome differences and identifying potential candidate genes affecting drip loss. RESULTS: As a result, 363 differentially expressed (DE) genes were detected in the comparative gene expression analysis, of which 239 were up-regulated and 124 were down-regulated in the low drip loss group. The DE genes were further filtered by correlation analysis between their expression and drip loss values in the 28 Duroc pigs measured and comparison of them with QTLs affecting drip loss. Consequently, of the 363 DE genes, 100 were identified as critical DE genes for drip loss. Functional analysis of these critical DE genes revealed some GO terms (extracellular matrix, cell adhesion mediated by integrin, heterotypic cell-cell adhesion), pathway (ECM-receptor interaction), and new potential candidate genes (TNC, ITGA5, ITGA11, THBS3 and CD44) which played an important role in regulating the variation of drip loss, and deserved to carry further studies to unravel their specific mechanism on drip loss. CONCLUSIONS: Our study revealed some GO terms, pathways and potential candidate genes affecting drip loss. It provides crucial information to understand the molecular mechanism of drip loss, and would be of help for improving meat quality of pigs.

MicroRNA Transcriptome of Poly I:C-Stimulated Peripheral Blood Mononuclear Cells Reveals Evidence for MicroRNAs in Regulating Host Response to RNA Viruses in Pigs.

MicroRNAs (miRNAs) are one family of small noncoding RNAs that function to modulate the activity of specific mRNA targets in animals. To understand the role of miRNAs in regulating genes involved in the host immune response to RNA viruses, we profiled and characterized the miRNAs of swine peripheral blood mononuclear cells (PBMC) stimulated with poly I:C, a synthetic dsRNA analog, by miRNA-sequencing (miRNA-seq). We identified a total of 905 miRNAs, of which 503 miRNAs were firstly exploited herein with no annotation in the latest miRBase 21.0. Expression analysis demonstrated that poly I:C stimulation can elicit significantly differentially expressed (DE) miRNAs in Dapulian (n = 20), one Chinese indigenous breed, as well as Landrace (n = 23). By integrating the mRNA expression profiles of the same sample with miRNA profiles, we carried out function analyses of the target genes of these DE miRNAs, with the results indicating that target genes were most enriched in some immune-related pathways and gene ontology (GO) terms, suggesting that DE miRNAs play an important role in the regulation of host to poly I:C stimulation. Furthermore, we also detected 43 and 61 significantly DE miRNAs between the two breeds in the control sample groups and poly I:C stimulation groups, respectively, which may be involved in regulation of the different characteristics of the two breeds. This study describes for the first time the PBMC miRNA transcriptomic response to poly I:C stimulation in pigs, which not only contributes to a broad view of the pig miRNAome but improves our understanding of miRNA function in regulating host immune response to RNA viruses.

Transcriptomic Analysis Identifies Candidate Genes and Gene Sets Controlling the Response of Porcine Peripheral Blood Mononuclear Cells to Poly I:C Stimulation.

Polyinosinic-polycytidylic acid (poly I:C), a synthetic dsRNA analog, has been demonstrated to have stimulatory effects similar to viral dsRNA. To gain deep knowledge of the host transcriptional response of pigs to poly I:C stimulation, in the present study, we cultured and stimulated peripheral blood mononuclear cells (PBMC) of piglets of one Chinese indigenous breed (Dapulian) and one modern commercial breed (Landrace) with poly I:C, and compared their transcriptional profiling using RNA-sequencing (RNA-seq). Our results indicated that poly I:C stimulation can elicit significantly differentially expressed (DE) genes in Dapulian (g = 290) as well as Landrace (g = 85). We also performed gene set analysis using the Gene Set Enrichment Analysis (GSEA) package, and identified some significantly enriched gene sets in Dapulian (g = 18) and Landrace (g = 21). Most of the shared DE genes and gene sets were immune-related, and may play crucial rules in the immune response of poly I:C stimulation. In addition, we detected large sets of significantly DE genes and enriched gene sets when comparing the gene expression profile between the two breeds, including control and poly I:C stimulation groups. Besides immune-related functions, some of the DE genes and gene sets between the two breeds were involved in development and growth of various tissues, which may be correlated with the different characteristics of the two breeds. The DE genes and gene sets detected herein provide crucial information towards understanding the immune regulation of antiviral responses, and the molecular mechanisms of different genetic resistance to viral infection, in modern and indigenous pigs.

Transcriptomic Analysis of the Porcine Endometrium during Embryo Implantation.

In pigs, successful embryo implantation is an important guarantee for producing litter size, and early embryonic loss occurring on day 12-30 of gestation critically affects the potential litter size. The implantation process is regulated by the expression of numerous genes, so comprehensive analysis of the endometrium is necessary. In this study, RNA sequencing (RNA-Seq) technology is used to analyze endometrial tissues during early pregnancy. We investigated the changes of gene expression between three stages (day 12, 18, and 25) by multiple comparisons. There were 1557, 8951, and 2345 differentially expressed genes (DEGs) revealed between the different periods of implantation. We selected several genes for validation by the use of quantitative real-time RT-PCR. Bioinformatic analysis of differentially expressed genes in the endometrium revealed a number of biological processes and pathways potentially involved in embryo implantation in the pig, most noticeably cell proliferation, regulation of immune response, interaction of cytokine-cytokine receptors, and cell adhesion. These results showed that specific gene expression patterns reflect the different functions of the endometrium in three stages (maternal recognition, conceptus attachment, and embryo implantation). This study identified comprehensive transcriptomic profile in the porcine endometrium and thus could be a foundation for targeted studies of genes and pathways potentially involved in abnormal endometrial receptivity and embryo loss in early pregnancy.

Gene expression analysis of porcine peripheral blood mononuclear cell in response to immune stimulation of Poly I:C.

Polyinosinic-polycytidylic acid (Poly I:C) is an analogue of natural double strand RNA (dsRNA), which can simulate the viral dsRNA and stimulate the immune response. In the present study, peripheral blood mononuclear cells (PBMC) were isolated from piglets of Dapulian and Landrace with different disease resistance, and stimulated with 20 µg/mL Poly I:C for 24 hours in vitro culture. The expression of several cytokines (IL6, IL8, TNFα, IL10, IRF3, IFNα and IFNγ) and three pattern recognition receptors (TLR3, TLR4 and RIG1) was determined by qRT-PCR. The results showed that, most of the cytokines or receptors had obvious expression change compared with the control (without Poly I:C stimulation), especially the three cytokine genes IL6, IL8 and IL10, whose average expression change times were 20.71, 10.87 and 5.18, respectively. Expression comparison between breeds and among individuals of the same breed indicated that there was obvious difference not only between Dapulain and Landrace (Dapulain higher than Landrace) but also among the three individuals of the same breed. Our study simulated the infection of dsRNA to host cells using Poly I:C, and provided experimental foundation for further study on selecting the immune genes in response to Poly I:C stimulation and identifying the unique disease-resistance genes of Dapulian.

Selection of Reference Genes for Gene Expression Studies in Porcine Whole Blood and Peripheral Blood Mononuclear Cells under Polyinosinic:Polycytidylic Acid Stimulation.

Investigating gene expression of immune cells of whole blood or peripheral blood mononuclear cells (PBMC) under polyinosinic:polycytidylic acid (poly I:C) stimulation is valuable for understanding the immune response of organism to RNA viruses. Quantitative real-time PCR (qRT-PCR) is a standard method for quantification of gene expression studies. However, the reliability of qRT-PCR data critically depends on proper selection of reference genes. In the study, using two different analysis programs, geNorm and NormFinder, we systematically evaluated the gene expression stability of six candidate reference genes (GAPDH, ACTB, B2M, RPL4, TBP, and PPIA) in samples of whole blood and PBMC with or without poly I:C stimulation. Generally, the six candidate genes performed a similar trend of expression stability in the samples of whole blood and PBMC, but more stably expressed in whole blood than in PBMC. geNorm ranked B2M and PPIA as the best combination for gene expression normalization, while according to NormFinder, TBP was ranked as the most stable reference gene, followed by B2M and PPIA. Comprehensively considering the results from the two programs, we recommended using the geometric mean of the three genes, TBP, PPIA and B2M, to normalize the gene expression of whole blood and PBMC with poly I:C stimulation. Our study is the first detailed survey of the gene expression stability in whole blood and PBMC with or without poly I:C stimulation and should be helpful for investigating the molecular mechanism involved in porcine whole blood and PBMC in response to poly I:C stimulation.

[Genomic DNA extraction from hair sacs of pigs using modified phenol-chloroform method].

In referring to various methods for genomic DNA extraction from different tissues, we modified the classical phenol-chloroform procedure and reaction system for use in genomic DNA extraction from pig hair sacs. With the modified the phenol-chloroform method we successfully obtained high quality genomic DNA from pig hair sacs. Genomic DNA can be extracted from sacs of one to six pig hairs with satisfied quantity and quality for the need of PCR-based molecular ex-periment.