Selection and Validation of Reference Genes for RT-qPCR Analysis in Tibetan Medicinal Plant Saussurea Laniceps Callus under Abiotic Stresses and Hormone Treatments.

Real-time quantitative PCR (RT-qPCR) is an important technique for studying gene expression analysis, but accurate and reliable results depend on the use of a stable reference gene. This study proposes to test the expression stability of candidate reference genes in the callus of Saussurea laniceps, a unique Tibetan medicinal plant. Based on the S. laniceps callus transcriptome, eleven candidate reference genes, including TUA2, TUB3, TUB8, TIF3B1, TIF3H1, ELF5A, PP2AA2, UEV1D, UBL5, UBC36, and SKIP1), were validated for RT-qPCR normalization in the callus under abiotic stress (salt, cold, and UV) and hormone treatments (abscisic acid, MeJA, and salicylic acid). The stability of the candidate genes was evaluated in all the samples of S. laniceps. Comprehensive analysis of all samples showed that the best reference genes were UBC36 and UBL5. ELF5A and TIF3B1 were ranked as the most stable genes in the sample sets under abiotic stress. For hormone stimulation, UBC36 and TIF3H1 genes had the best stability. This study provides useful guidelines and a starting point for reference gene selection for expression analysis using RT-qPCR techniques in S. laniceps.

[Thermal desorption sampling-gas chromatography-tandem mass spectrometry with a carbonyl-iron powder composite silica monolithic column for enrichment and determination of pyrethroid pesticide residues in tea samples].

Long-term exposure to pyrethroid insecticides is detrimental to the nervous system, reproductive system, and immune system in humans. Therefore, enrichment detection of pyrethroid pesticides is imperative. In this study, a novel carbonyl-iron powder composite silica monolithic column was first prepared for the enrichment of pyrethroid pesticide residues in tea samples. Then, the target analytes were thermally desorbed and online-injected into a gas chromatography-tandem mass spectrometry (GC-MS/MS) system. In the present method, hydroxy-terminated polydimethylsiloxane (PDMS) was covalently bonded to the surface of the SiO2 network and subsequently bonded with the carbonyl-iron powder. After the target analytes were adsorbed and concentrated in the PDMS spots, high-frequency induction heating was used for GC-MS/MS sampling. Under the optimal conditions, the detection limits of the pyrethroid pesticide residues were 3.8 to 7.5 µg/kg, and the relative standard deviation was 3.2% to 6.8% (n=6). The extraction recovery ranged from 97.7% to 110.5%, and the correlation coefficient was ≥ 0.9960. In addition, the enrichment factor could reach 1000 times. PDMS materials show excellent adsorption properties for non-polar solutes. In our experiment, carbonyl iron powder-bonded monolithic columns were prepared on the basis of stir bar sorptive extraction (SBSE). Carbonyl iron powder magnetic particles were evenly implanted into the inorganic-organic hybrid cassia material for realizing rapid and uniform desorption upon electromagnetic induction heating. Under the premise of perfectly integrating the technical advantages of SBSE and solid-phase microextraction (SPME), the electromagnetic induction characteristics of carbonyl iron powder can be exploited for thermal desorption and directly combined with GC-MS to facilitate online analysis and solvent-free elution. Compared with the conventional SPME method, the proposed method has the advantages of high enrichment factor, large adsorption capacity of the column, reusability, high degree of automation, and good universality. This method has high significance for sample preparation and for the extraction of pesticide residues in complex matrices.

[Rapid determination of 204 pesticide residues in tea by ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry].

An analytical method was established for the simultaneous determination of 204 pesticide residues in tea by ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS). The samples were extracted with acetonitrile, and cleaned-up by solid phase extraction (SPE) with a Carb-PSA cartridge, eluted with acetonitrile-toluene (3:1, v/v), determined by UPLC-Q-TOF/MS and quantified by external standard method. A data base of the accurate mass numbers and a library which contains the 204 pesticides were established. The automatic retrieval of detection results was carried on according to the characteristics of the compound, such as accurate mass, retention time, isotopic ratio, and so on. Based on the above results, the qualitative identifications of the 204 pesticides were accomplished without the contrast of standard substances. The results indicated that this method can be used to determine the 204 pesticides in tea. At the three spiked levels of 10, 20, 50 µg/kg, mean recoveries for the 204 pesticides in tea were between 68. 1% and 117. 2%, with the relative standard deviations (RSDs) ranging from 3.1% to 18.9%. The limits of quantification for the 204 pesticides were lower than 10 µg/kg. The method has been applied to four positive samples, and the results generally accord with the detection results by the method of GB/T 23205-2008. This method has the characteristics of high efficiency, as well as high sensitivity and accuracy, which can meet the requirement of the determination of the 204 pesticides in tea.