Selection and Validation of Reference Genes for Gene Expression in Bactericera gobica Loginova under Different Insecticide Stresses.

Insecticide resistance has long been a problem in crop pest control. Bactericera gobica is a major pest on the well-known medicinal plants Lycium barbarum L. Investigating insecticide resistance mechanisms of B. gobica will help to identify pesticide reduction strategies to control the pest. Gene expression normalization by RT-qPCR requires the selection and validation of appropriate reference genes (RGs). Here, 15 candidate RGs were selected from transcriptome data of B. gobica. Their expression stability was evaluated with five algorithms (Delta Ct, GeNorm, Normfinder, BestKeeper and RefFinder) for sample types differing in response to five insecticide stresses and in four other experimental conditions. Our results indicated that the RGs RPL10 + RPS15 for Imidacloprid and Abamectin; RPL10 + AK for Thiamethoxam; RPL32 + RPL10 for λ-cyhalothrin; RPL10 + RPL8 for Matrine; and EF2 + RPL32 under different insecticide stresses were the most suitable RGs for RT-qPCR normalization. EF1α + RPL8, EF1α + ß-actin, ß-actin + EF2 and ß-actin + RPS15 were the optimal combination of RGs under odor stimulation, temperature, developmental stages and both sexes, respectively. Overall, EF2 and RPL8 were the two most stable RGs in all conditions, while α-TUB and RPL32 were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target cytochrome P450 CYP6a1 gene between adult and nymph stages and under imidacloprid stress. The results of CYP6a1 expression were consistent with transcriptome data. This study is the first research on the most stable RG selection in B. gobica nymphs exposed to different insecticides, which will contribute to further research on insecticide resistance mechanisms in B. gobica.

Selection and validation of reference genes for RT-qPCR in ophiocordyceps sinensis under different experimental conditions.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (ß-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. ß-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.

[Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii].

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species.

Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans.

The quantitative polymerase chain reaction (qRT-PCR) technique gives promising opportunities to detect and quantify RNA targets and is commonly used in many research fields. This study aimed to identify suitable reference genes for physical exercise and omega-3 fatty acids supplementation intervention. Forty healthy, physically active men were exposed to a 12-week eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation and standardized endurance training protocol. Blood samples were collected before and after the intervention and mRNA levels of six potential reference genes were tested in the leukocytes of 18 eligible participants using the qRT-PCR method: GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), ACTB (Beta actin), TUBB (Tubulin Beta Class I), RPS18 (Ribosomal Protein S18), UBE2D2 (Ubiquitin-conjugating enzyme E2 D2), and HPRT1 (Hypoxanthine Phosphoribosyltransferase 1). The raw quantification cycle (Cq) values were then analyzed using RefFinder, an online tool that incorporates four different algorithms: NormFinder, geNorm, BestKeeper, and the comparative delta-Ct method. Delta-Ct, NormFinder, BestKeeper, and RefFinder comprehensive ranking have found GAPDH to be the most stably expressed gene. geNorm has identified TUBB and HPRT as the most stable genes. All algorithms have found ACTB to be the least stably expressed gene. A combination of the three most stably expressed genes, namely GAPDH, TUBB, and HPRT, is suggested for obtaining the most reliable results.

Comparison between droplet digital PCR and reverse transcription-quantitative PCR methods to measure ecotoxicology biomarkers.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is currently the gold-standard technique for detecting and quantifying messenger RNA. However, without proper validation, the method may produce artefactual and non-reproducible cycle threshold values generating poor-quality data. The newer droplet digital PCR (ddPCR) method allows for the absolute quantification of targeted nucleic acids providing more sensitive and accurate measurements without requiring external standards. This study compared these two PCR-based methods to measure the expression of well-documented genes used in ecotoxicology studies. We exposed Mediterranean mussels (Mytilus galloprovincialis) to copper and analyzed gene expression in gills and digestive glands using RT-qPCR and ddPCR assays. A step-by-step methodology to optimize and compare the two technologies is described. After ten-fold serial complementary DNA dilution, both RT-qPCR and ddPCR exhibited comparable linearity and efficiency and produced statistically similar results. We conclude that ddPCR is a suitable method to assess gene expression in an ecotoxicological context. However, RT-qPCR has a shorter processing time and remains more cost-effective.

Reducing COVID-19 diagnostic errors with dNTPαSe supplementation.

Timely and accurate diagnosis of COVID-19 is critical for controlling the pandemic. As the standard method to diagnose SARS-CoV-2, the real-time reverse transcription polymerase chain reaction (RT-qPCR) has good convenience. However, RT-qPCR still has a relatively high false-negative rate, particularly in the case of detecting low viral loads. In this study, using selenium-modified nucleoside triphosphates (dNTPαSe) in the RT-PCR reactions, we successfully increased the detection sensitivity and reduced the false-negative rate in COVID-19 diagnosis. By detecting positive controls, pseudovirus, and clinical samples with the commercial kits, we found that the dNTPαSe supplementation to these kits could generally offer smaller Ct values, permit the viral detection even in single-digit copies, and increase the detection specificity, sensitivity, and accuracy, thereby reducing the false-negative rate. Our experimental results demonstrated that dNTPαSe supplementation can make the commercial kits more specific, sensitive, and accurate, and this method is a convenient and efficient strategy for the disease detection and diagnosis.

Validation of suitable reference genes by various algorithms for gene expression analysis in Isodon rubescens under different abiotic stresses.

Isodon rubescens (Hemsley) H. Hara (Lamiaceae) is a traditional Chinese medicine plant that has been used to treat various human diseases. Oridonin is one of the main active ingredients, and the route of its molecular biosynthesis remains to be determined. The study of gene expression patterns can provide clues toward the understanding of its biological functions. The selection of suitable reference genes for normalizing target gene expression is the first steps in any quantitative real-time PCR (RT-qPCR) gene expression study. Therefore, validation of suitable reference genes is necessary for obtaining reliable results in RT-qPCR analyses of I. rubescens. Here, 12 candidate reference genes were chosen, and their expression stability in different tissues of I. rubescens and in leaves under different abiotic stresses (NaCl, dehydration, SA, MeJA, and ABA) was evaluated using the ∆Ct, NormFinder, GeNorm, BestKeeper, and RankAggreg statistical tools. Analysis using the comprehensive tools of RankAggreg algorithm showed that GADPH, 18S and eIF were stably expressed in different tissues; UBQ, Apt, and HIS; Cycl, UBQ, and PP2A; GADPH, 18S, and eIF; eIF, UBQ, and PP2A; TUB, Cycl, and UBQ; were the best three candidate reference genes for the samples of Dehydration, NaCl, SA, MeJA, and ABA treatment, respectively. While for the concatenated sets of ND (NaCl and dehydration) and SMA (SA, MeJA, and ABA), UBQ, HIS, and TUA; UBQ, eIF and Apt were the three appropriate candidate reference genes, respectively. In addition, the expression patterns of HMGR in different tissues and under different treatments were used to confirm the reliability of the selected reference genes, indicating that the use of an inappropriate reference gene as the internal control will cause results with a large deviation. This work is the first study on the expression stability of reference genes in I. rubescens and will be particularly useful for gene functional research in this species.

Systematic Identification and Validation of Suitable Reference Genes for the Normalization of Gene Expression in Prunella vulgaris under Different Organs and Spike Development Stages.

The quantitative real-time PCR (qRT-PCR) is an efficient and sensitive method for determining gene expression levels, but the accuracy of the results substantially depends on the stability of the reference gene (RG). Therefore, choosing an appropriate reference gene is a critical step in normalizing qRT-PCR data. Prunella vulgaris L. is a traditional Chinese medicine herb widely used in China. Its main medicinal part is the fruiting spike which is termed Spica Prunellae. However, thus far, few studies have been conducted on the mechanism of Spica Prunellae development. Meanwhile, no reliable RGs have been reported in P. vulgaris. The expression levels of 14 candidate RGs were analyzed in this study in various organs and at different stages of Spica Prunellae development. Four statistical algorithms (Delta Ct, BestKeeper, NormFinder, and geNorm) were utilized to identify the RGs' stability, and an integrated stability rating was generated via the RefFinder website online. The final ranking results revealed that eIF-2 was the most stable RG, whereas VAB2 was the least suitable as an RG. Furthermore, eIF-2 + Histon3.3 was identified as the best RG combination in different periods and the total samples. Finally, the expressions of the PvTAT and Pv4CL2 genes related to the regulation of rosmarinic acid synthesis in different organs were used to verify the stable and unstable RGs. The stable RGs in P. vulgaris were originally identified and verified in this work. This achievement provides strong support for obtaining a reliable qPCR analysis and lays the foundation for in-depth research on the developmental mechanism of Spica Prunellae.

Selection of reference genes for quantitative real-time PCR normalization in the coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae).

The coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is a major destructive pest of Coffea arabica L. (Gentianales: Rubiaceae), widely planted in many Asian countries, including China. Quantitative real-time polymerase chain reaction (qRT-PCR) is a common method for quantitative analysis of gene transcription levels. To obtain accurate and reliable qRT-PCR results, it is necessary to select suitable reference genes to different experimental conditions for normalizing the target gene expression. However, the stability of the expression of reference genes in X. quadripes has rarely been studied. In this study, the expression stability of nine candidate reference genes were investigated under biotic and abiotic conditions for use in qRT-PCR's normalization. By integrating the results of four algorithms of NormFinder, BestKeeper, geNorm, and RefFinder, the optimal reference gene combinations in different experimental conditions were performed as follows: RPL10a and EIF3D were the optimal reference genes for developmental stage samples, EIF4E, RPL10a, and RPS27a for tissue samples, V-ATP and EF1α for the sex samples, EIF3D and V-ATP for temperature treatment, RPS27a and RPL10a for insecticide stress, and RPL10a, RPS27a, and EF1α for all the samples. This study will help to obtain the stable internal reference genes under biotic and abiotic conditions and lay the foundation for in-depth functional research of target genes or genomics on olfactory molecular mechanisms, temperature adaptability, and insecticide resistance in X. quadripes.

Proteomics-based screening and antibiotic resistance assessment of clinical and sub-clinical Brucella species: An evolution of brucellosis infection control.

Brucellae are intracellular sneaky bacteria and they can elude the host's defensive mechanisms, resulting in therapeutic failure. Therefore, the goal of this investigation was to rapid identification of Brucella species collected from animals and humans in Saudi Arabia, as well as to evaluate their resistance to antibiotics. On selective media, 364 animal samples as well as 70 human blood samples were cultured. Serological and biochemical approaches were initially used to identify a total of 25 probable cultured isolates. The proteomics of Brucella species were identified using the MALDI Biotyper (MBT) system, which was subsequently verified using real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Both Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) were tested for antimicrobial susceptibility using Kirby Bauer method and the E-test. In total, 25 samples were positive for Brucella and included 11 B. melitensis and 14 B. abortus isolates. Twenty-two out of 25 (88%) and 24/25 (96%) of Brucella strains were recognized through the Vitek 2 Compact system. While MBT was magnificently identified 100% of the strains at the species level with a score value more than or equal to 2.00. Trimethoprim-sulfamethoxazole, rifampin, ampicillin-sulbactam, and ampicillin resistance in B. melitensis was 36.36%, 31.82%, 27.27%, and 22.70%, respectively. Rifampin, trimethoprim-sulfamethoxazole, ampicillin, and ampicillin-sulbactam resistance was found in 35.71%, 32.14%, 32.14%, and 28.57% of B. abortus isolates, correspondingly. MBT confirmed by microfluidic electrophoresis is a successful approach for identifying Brucella species at the species level. The resistance of B. melitensis and B. abortus to various antibiotics should be investigated in future studies.

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression in Goji Fruit Fly (Neoceratitis asiatica Becker) under Developmental Stages and Five Abiotic Stresses.

Goji fruit fly, Neoceratitis asiatica, is a major pest on the well-known medicinal plant Lycium barbarum. Dissecting molecular mechanisms of infestation and host selection of N. asiatica will contribute to the determination of best management practices for pest fly control. Gene expression normalization by Real-time quantitative PCR (qPCR) requires the selection and validation of appropriate reference genes (RGs). Hence, 15 candidate RGs were selected from transcriptome data of N. asiatica. Their expression stability was evaluated with five algorithms (∆Ct, Normfinder, GeNorm, BestKeeper, and RefFinder) for sample types differing in the developmental stage, sex, tissue type, and in response to five different abiotic stresses. Our results indicated that the RGs ß-Actin + GST for sex, RPL32 + EF1α for tissue type, RPS13+ EF1α for developmental stages along with odor stimulation, color induction, and starvation-refeeding stresses, EF1α + GAPDH under insecticide stress, RPS13 + RPS18 under temperature stress, respectively, were selected as the most suitable RGs for qPCR normalization. Overall, RPS18 and EF1α were the two most stable RGs in all conditions, while RPS15 and EF1ß were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target odorant-binding protein OBP56a gene in male and female antennae, different tissues, and under odor stimulation. The results of OBP56a expression were consistent with transcriptome data. Our study is the first research on the most stable RGs selection in N. asiatica, which will facilitate further studies on the mechanisms of host selection and insecticide resistance in N. asiatica.

Use of Molecular Methods to Authenticate Animal Species and Tissue in Bovine Liver Dietary Supplements.

Dietary supplements containing bovine (subfamily Bovinae) liver are susceptible to fraud due to their high value and the lack of modern detection methods available for processed animal tissues. The objective of this research was to use molecular methods to authenticate dietary supplements claiming to contain bovine liver or beef liver through the verification of animal species and tissue type. A total of 53 bovine/beef liver dietary supplements were purchased from online sources. The presence of liver was verified with reverse transcription and real-time PCR testing for microRNA-122 (miR-122), which is highly expressed in liver tissue. Multiplex real-time PCR targeting domestic cattle (Bos taurus), horse (Equus caballus), sheep (Ovis aries), and pork (Sus scrofa) was used to verify species. Samples that failed species identification with multiplex real-time PCR underwent DNA mini-barcoding. Overall, bovine species were detected in 48/53 liver supplements: 35 samples were confirmed as domestic cattle with multiplex real-time PCR and an additional 13 samples were confirmed as domestic cattle or Bos spp. with DNA mini-barcoding. One of these samples was also positive for sheep/lamb, which was declared on the label. One product contained undeclared pork in addition to beef. MiR-122 was detected in 51 out of 53 supplements, suggesting the presence of liver. While this study demonstrates the potential use of tissue-specific microRNAs in verifying tissues in dietary supplements, more research is needed to evaluate the specificity of these markers.

A Long-Amplicon Viability-qPCR Test for Quantifying Living Pathogens that Cause Bacterial Spot in Tomato Seed.

Bacterial spot is one of the most serious diseases of tomato. It is caused by four species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated or infected seed can be a major source of inoculum for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. Current seed testing protocols rely mainly on plating the seed extract and conventional PCR; however, the plating method cannot detect viable but nonculturable cells, and the conventional PCR assay has limited capability to differentiate DNA extracted from viable or dead bacterial cells. To improve the sensitivity and specificity of the tomato seed testing method for bacterial spot pathogens, a long-amplicon quantitative PCR (qPCR) assay coupled with propidium monoazide (PMA-qPCR) was developed to quantify selectively the four pathogenic Xanthomonas species in tomato seed. The optimized PMA-qPCR procedure was evaluated on pure bacterial suspensions, bacteria-spiked seed extracts, and seed extracts of inoculated and naturally infected seed. A crude DNA extraction protocol also was developed, and PMA-qPCR with crude bacterial DNA extracts resulted in accurate quantification of 104 to 108 CFU/ml of viable bacteria when mixed with dead cells at concentrations as high as 107 CFU/ml in the seed extracts. With DNA purified from concentrated seed extracts, the PMA-qPCR assay was able to detect DNA of the target pathogens in seed samples spiked with ≥75 CFU/ml (about 0.5 CFU/seed) of the viable pathogens. Latent class analysis of the inoculated and naturally infected seed samples showed that the PMA-qPCR assay had greater sensitivity than plating the seed extracts on the semiselective modified Tween Medium B and CKTM media for all four target species. Being much faster and more sensitive than dilution plating, the PMA-qPCR assay has potential to be used as a standalone tool or in combination with the plating method to improve tomato seed testing and advance the production of clean seed.

A real-time recombinase polymerase amplification assay for fast and accurate detection of Ditylenchus destructor.

Ditylenchus destructor is a plant-parasitic nematode that seriously infests sweet potato crop in China. Thus, fast and accurate detection of D. destructor in soil and plant tissue samples is of great significance. In this study, a real-time recombinase polymerase amplification (RPA) assay was developed for the rapid and accurate detection of D. destructor in various samples. The RPA assay could be easily operated and detected as low as 1/500 individual J4 nematode DNA per reaction in 20 min at 39 °C with high specificity. The assay meets the requirements of rapid detection prior to port quarantine as well as on-site real-time detection and can be applied to detect the parasite in soil and plant samples. The modified gDNA extraction method for a single nematode established in this study significantly reduced the time of detection and improved the applicability of the real-time RPA assay for on-site detection in different environments. The real-time RPA assay to detect D. destructor will be useful for epidemiological investigations in the field as well as for quarantine processes in the sweet potato and potato trade.

Identification of reliable reference genes for quantitative real-time PCR analysis of the Rhus chinensis Mill. leaf response to temperature changes.

Rhus chinensis Mill. (RCM) is the host plant of Galla chinensis, which is valued in traditional medicine. Environmental temperature directly determines the probability of gallnut formation and RCM growth. At present, there is no experiment to systematically analyse the stability of internal reference gene (RG) expression in RCM. In this experiment, leaves that did not form gallnuts were used as the control group, while leaves that formed gallnuts were used as the experimental group. First, we conducted transcriptome experiments on RCM leaves to obtain 45 103 differential genes and functional enrichment annotations between the two groups. On this basis, this experiment established a transcriptional gene change model of leaves in the process of gallnut formation after being bitten by aphids, and RCM reference candidate genes were screened from RNA sequencing (RNA-seq) data. This study is based on RCM transcriptome data and evaluates the stability of 11 potential reference genes under cold stress (4 °C) and heat stress (34 °C), using three statistical algorithms (geNorm, NormFinder, and BestKeeper). The results show that GAPDH1 + PP2A2/UBQ are stable reference genes under heat stress, while GAPDH1 + ACT are the most stable under cold stress. This study is the first to screen candidate reference genes in RCM and could help guide future molecular studies in this genus.

Synthetic Biology Medicine and Bacteria-Based Cancer Therapeutics.

Spontaneous tumor regression following bacterial infection has been observed for hundreds of years. These observations along with anecdotal medical findings in 1890s led to the development of Coley's "toxins," consisting of killed Streptococcus pyogenes and Serratia marcescens bacteria, as the first cancer immunotherapy. The use of this approach, however, was not widely accepted at the time especially after the introduction of radiation therapy as a treatment for cancer in the early 1900s. Over the last 30-40 years there has been renewed interest in the use of bacteria to treat human solid tumors. This is based on the observation that various nonpathogenic anaerobic bacteria can infiltrate and replicate within solid tumors when given intravenously. Bacteria tested as potential anticancer agents include the Gram-positive obligate anaerobes Bifidobacterium and Clostridium, as well as the gram-negative facultative anaerobe Salmonella. Recent advances in synthetic biology and clinical success in cancer immunotherapy provide renewed momentum for developing bacteria-based cancer immunotherapy for cancer treatment and should allow greater potential for the development of novel therapeutic approaches for this devastating disease.

Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca's response to salinity.

Quantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

Quantification of Phytophthora infestans population densities and their changes in potato field soil using real-time PCR.

Tuber infection of Phytophthora infestans often occurs at harvest. However, it is difficult to accurately estimate the population densities of P. infestans in soil, especially Japanese soil. In the present study, P. infestans DNA was extracted from soil samples using a modified CTAB-bead method and quantified using real-time PCR to accurately, rapidly and easily estimate the P. infestans population densities in upland soils in Japan. P. infestans was well quantified in eleven types of soil samples, including nine types of upland soils in Japan, that were artificially inoculated with a zoosporangia suspension. The amounts of P. infestans DNA estimated by the real-time PCR were proportional to the inoculum densities. In the non-controlled experimental potato field, P. infestans population densities in soil corresponded to the development of symptoms and were correlated with the number of lesions on the potato foliage. These results imply that the proposed real-time PCR assay is suitable for the estimation or monitoring of P. infestans population densities in upland soils in Japan. The population densities at the ridge bottoms were larger than those at any other location in commercial potato fields. These results were similar to those of a previous report using a bioassay. Moreover, a correlation between DNA quantity and inoculum potential was observed. In conclusion, the real-time PCR assay developed in this study is suitable for indirect estimation of the inoculum potential of P. infestans.

Estimating included animal species in mixed crude drugs derived from animals using massively parallel sequencing.

We developed a method that can detect each animal species of origin for crude drugs derived from multiple animal species based on massively parallel sequencing analysis of mitochondrial genes. The crude drugs derived from animals investigated in this study were Cervi Parvum Cornu and Trogopterorum feces, which are derived from a mix of different animal species, two chopped cicada sloughs, and two commercial Kampo drugs. The mitochondrial 12S rRNA, 16S rRNA, and cytochrome oxidase subunit I gene regions were amplified and sequenced using MiSeq. The ratios of haplotype to total number of sequences reads were calculated after sequence extraction and trimming. Haplotypes that exceeded the threshold were defined as positive haplotypes, which were compared with all available sequences using BLAST. In the Cervi Parvum Cornu and Trogopterorum feces samples, the haplotype ratios corresponded roughly to the mixture ratios, although there was a slight difference from mixture ratios depending on the gene examined. This method could also roughly estimate the compositions of chopped cicada sloughs and Kampo drugs. This analysis, whereby the sequences of several genes are elucidated, is better for identifying the included animal species. This method should be useful for quality control of crude drugs and Kampo drugs.