An RT-PCR based microchip test system for the detection of SARS-CoV-2 offers pre-loaded and lyophilized reagents in the microchip. However, the 30- and 48-microwell formats of the microchip being miniaturized and perf...An RT-PCR based microchip test system for the detection of SARS-CoV-2 offers pre-loaded and lyophilized reagents in the microchip. However, the 30- and 48-microwell formats of the microchip being miniaturized and performing 1.2 μl reaction, seek visual attention during sample addition. Therefore, adding colorants as color indicator in the lyophilized matrix in the microchips or adding to sample or master mix can impart not only user-friendliness to the task of liquid handling but also precision, and color-codes for easy identification of multiple kits in the layout of the microchip without compromising PCR data quality. A panel of colorants was screened for their background intensity, spectral inertness towards detection channels of AriaDNA<sup>TM</sup> analyzer, interference with the reporter dyes (FAM, Cy5 and ROX), and visibility of optimal concentration in the microwell. The concentration of the colorant displaying insignificant impact on the quality of the amplification (Ct, fluorescence, and sensitivity) in comparison to no-colorant control was chosen for inclusion in the test kit. Tartrazine, Acid Red, Brilliant Blue and FAST Green colorants lyophilized with the reagents in the SARS-CoV-2 microchips were found to be stable and suitable. Storage of microchips with Fast Green colorant was tested at 40°C, 22<span style="white-space:normal;">°</span>C, 4<span style="white-space:normal;">°</span>C, and -20<span style="white-space:normal;">°</span>C for 70 days and was found to be suitable and compatible with different master mixes available as liquid or lyophilized. Additionally, the microchips pre-loaded with lyophilized reagents in the presence and absence of two colorants Tartrazine and Fast Green were validated with clinical samples of SARS-COV-2. No significant impact of these colorants both intra- and inter-microchips was observed on the Ct and intensity of amplification for the tested samples in comparison to no-colorant control. The data suggested that the tested colorants can be used to color the sample, or the master mix or PCR mix for user-friendly liquid handling in empty microchips. For the microchip with pre-loaded and lyophilized reagents, the colorant can be added to lyophilized mixture for precision liquid handling and color-coding of lyophilized kits in the microchips. The manufacturing quality of the lyophilized microchips can also improve with colorant loaded reagent mix.展开更多
The recombinant plasmid pFV2cfGH was constructed using Growth hormone gene which was cloned from the channel catfish cDNA library,the β-action promoter of carp and the polyadenylation signal from the salmon growth ho...The recombinant plasmid pFV2cfGH was constructed using Growth hormone gene which was cloned from the channel catfish cDNA library,the β-action promoter of carp and the polyadenylation signal from the salmon growth hormone gene, and recombinant plasmid pFV2cfGH was microinjected into the fertilized eggs of the colorful crucian carp. 60 transgenic Colorful Crucian carps total DNA were got ,and growth hormone gene were detected by PCR. The result shows that the channel catfish growth hormone gene had been transmitted into the genome of the Colorful Crucian carp and the integration rate is about 47%.展开更多
Pigmentation plays important adaptation and physiological efficiency roles in animals. In the sequence of a 648 bp fragment representing intron 1, exon 2, and part of intron 2 of the MLPH mammalian pigmentation gene, ...Pigmentation plays important adaptation and physiological efficiency roles in animals. In the sequence of a 648 bp fragment representing intron 1, exon 2, and part of intron 2 of the MLPH mammalian pigmentation gene, we identified a novel g.469C> G mutation in intron 2, and genotyped it in 266 Nigerian goats using PCR-RFLP analysis. The C allele had frequencies of 0.9625, 0.9804 and 0.97405 in West African Dwarf (WAD),Sahel(SH) and Red Sokoto (RS) breeds, respectively. The G allele was the highest in WAD (0.0375), followed by RS (0.02595), and then SH (0.0196). Overall low FIS and FST and high Nm values demonstrate little differentiation within and among the goat breeds at this intronic locus. This g.469C> G polymorphism in MLPH gene is the first in any goat breed and also first in Nigerian goats. Our results suggest that this intronic SNP locus is maintained at Hardy-Weinberg equilibrium (P < 0.05) and the lack of association of this SNP with coat color may indicate its neutrality in goats.展开更多
文摘An RT-PCR based microchip test system for the detection of SARS-CoV-2 offers pre-loaded and lyophilized reagents in the microchip. However, the 30- and 48-microwell formats of the microchip being miniaturized and performing 1.2 μl reaction, seek visual attention during sample addition. Therefore, adding colorants as color indicator in the lyophilized matrix in the microchips or adding to sample or master mix can impart not only user-friendliness to the task of liquid handling but also precision, and color-codes for easy identification of multiple kits in the layout of the microchip without compromising PCR data quality. A panel of colorants was screened for their background intensity, spectral inertness towards detection channels of AriaDNA<sup>TM</sup> analyzer, interference with the reporter dyes (FAM, Cy5 and ROX), and visibility of optimal concentration in the microwell. The concentration of the colorant displaying insignificant impact on the quality of the amplification (Ct, fluorescence, and sensitivity) in comparison to no-colorant control was chosen for inclusion in the test kit. Tartrazine, Acid Red, Brilliant Blue and FAST Green colorants lyophilized with the reagents in the SARS-CoV-2 microchips were found to be stable and suitable. Storage of microchips with Fast Green colorant was tested at 40°C, 22<span style="white-space:normal;">°</span>C, 4<span style="white-space:normal;">°</span>C, and -20<span style="white-space:normal;">°</span>C for 70 days and was found to be suitable and compatible with different master mixes available as liquid or lyophilized. Additionally, the microchips pre-loaded with lyophilized reagents in the presence and absence of two colorants Tartrazine and Fast Green were validated with clinical samples of SARS-COV-2. No significant impact of these colorants both intra- and inter-microchips was observed on the Ct and intensity of amplification for the tested samples in comparison to no-colorant control. The data suggested that the tested colorants can be used to color the sample, or the master mix or PCR mix for user-friendly liquid handling in empty microchips. For the microchip with pre-loaded and lyophilized reagents, the colorant can be added to lyophilized mixture for precision liquid handling and color-coding of lyophilized kits in the microchips. The manufacturing quality of the lyophilized microchips can also improve with colorant loaded reagent mix.
基金Supported by Natural Science Foundation of Inner Mongolia Autonomous Region under Grant(No.2013MS1178 and No.2012MS1165)Science and Technology Innovation Guiding the Fund Project of Inner Mongolia Autonomous Region “The construction of Brucella infection early diagnosis and natural infection and artificial immune identification Kit”The Joint program of Tongliao City and Inner Mongolia University for the Nationalities under Grant(No.SXZD2012019)~~
文摘The recombinant plasmid pFV2cfGH was constructed using Growth hormone gene which was cloned from the channel catfish cDNA library,the β-action promoter of carp and the polyadenylation signal from the salmon growth hormone gene, and recombinant plasmid pFV2cfGH was microinjected into the fertilized eggs of the colorful crucian carp. 60 transgenic Colorful Crucian carps total DNA were got ,and growth hormone gene were detected by PCR. The result shows that the channel catfish growth hormone gene had been transmitted into the genome of the Colorful Crucian carp and the integration rate is about 47%.
文摘Pigmentation plays important adaptation and physiological efficiency roles in animals. In the sequence of a 648 bp fragment representing intron 1, exon 2, and part of intron 2 of the MLPH mammalian pigmentation gene, we identified a novel g.469C> G mutation in intron 2, and genotyped it in 266 Nigerian goats using PCR-RFLP analysis. The C allele had frequencies of 0.9625, 0.9804 and 0.97405 in West African Dwarf (WAD),Sahel(SH) and Red Sokoto (RS) breeds, respectively. The G allele was the highest in WAD (0.0375), followed by RS (0.02595), and then SH (0.0196). Overall low FIS and FST and high Nm values demonstrate little differentiation within and among the goat breeds at this intronic locus. This g.469C> G polymorphism in MLPH gene is the first in any goat breed and also first in Nigerian goats. Our results suggest that this intronic SNP locus is maintained at Hardy-Weinberg equilibrium (P < 0.05) and the lack of association of this SNP with coat color may indicate its neutrality in goats.
