Rapid and convenient quantitative analysis of SARS-CoV-2 RNA in serous saliva with a direct PCR method

Sensitive and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), frequently performed using direct polymerase chain reaction (PCR), is essential for restricting the spread of coronavirus disease 2019 (COVID-19). However, studies evaluating accurate detection are still required. This study evaluated the quantitativeness and sensitivity of the Ampdirect™ 2019-nCoV detection kit, a direct PCR method. Using saliva with or without Tris-buffered saline (TBS) dilution, linearity, and limits of the N1 and N2 regions of SARS-CoV-2 genomic RNA were assessed using EDX SARS-CoV-2 RNA standard dissolved in RNase-free water (RFW). Fluorescence intensities in non-diluted saliva were higher than those in TBS-diluted samples. Linear regression analysis of detected quantification cycle values and spiked standard RNA concentrations showed that the coefficient of determination of the N1 and N2 genes was 0.972 and 0.615 in RFW and 0.947 and 0.660 in saliva, respectively. N1- and N2-positive detection rates in saliva were 46% (6/13 tests) and 0% (0/12 tests) at one copy/reaction, respectively. These results indicate good quantitativeness and sensitivity for N1 but not for N2. Therefore, our findings reveal that the Ampdirect™ 2019-nCoV system, especially targeting the N1 gene, enables rapid and convenient quantification of SARS-CoV-2 RNA in saliva at one copy/reaction

Microarray Analysis of Perinatal-Estrogen-Induced Changes in Gene Expression Related to Brain Sexual Differentiation in Mice

<div><p>Sexual dimorphism of the behaviors or physiological functions in mammals is mainly due to the sex difference of the brain. A number of studies have suggested that the brain is masculinized or defeminized by estradiol converted from testicular androgens in perinatal period in rodents. However, the mechanisms of estrogen action resulting in masculinization/defeminization of the brain have not been clarified yet. The large-scale analysis with microarray in the present study is an attempt to obtain the candidate gene(s) mediating the perinatal estrogen effect causing the brain sexual differentiation. Female mice were injected with estradiol benzoate (EB) or vehicle on the day of birth, and the hypothalamus was collected at either 1, 3, 6, 12, or 24 h after the EB injection. More than one hundred genes down-regulated by the EB treatment in a biphasic manner peaked at 3 h and 12-24 h after the EB treatment, while forty to seventy genes were constantly up-regulated after it. Twelve genes, including <i>Ptgds, Hcrt</i>, <i>Tmed2</i>, <i>Klc1</i>, and <i>Nedd4</i>, whose mRNA expressions were down-regulated by the neonatal EB treatment, were chosen for further examination by semiquantitative RT-PCR in the hypothalamus of perinatal intact male and female mice. We selected the genes based on the known profiles of their potential roles in brain development. mRNA expression levels of <i>Ptgds, Hcrt</i>, <i>Tmed2</i>, and <i>Nedd4</i> were significantly lower in male mice than females at the day of birth, suggesting that the genes are down-regulated by estrogen converted from testicular androgen in perinatal male mice. Some genes, such as <i>Ptgds</i> encoding prostaglandin D2 production enzyme and <i>Hcrt</i> encording orexin, have been reported to have a role in neuroprotection. Thus, <i>Ptgds</i> and <i>Hcrt</i> could be possible candidate genes, which may mediate the effect of perinatal estrogen responsible for brain sexual differentiation. </p> </div

Changes in expressions of <i>Tmed2</i>, <i>Klc1</i>, <i>Nedd4, Hcrt</i>, <i>Meis2</i>, <i>Neurod6</i>, <i>Pitx2, Ptgds</i>, <i>Vtn</i>, Fn <i>1</i>, <i>Apod</i>, and <i>Igf2</i> genes in the female mouse hypothalamus 3 h (A), 12 h (B), or 24 h (C) after the EB treatment.

<p>Values in EB-treated (solid circle) and vehicle-treated controls (open circle) are indicated as signal intensity in microarray hybridization. Values are means±SEM. Values marked with asterisks (* or **) are significantly different from vehicle-treated controls at each time point (P < 0.05 or P < 0.01, two-way ANOVA (treatment and time as main factors) followed by the Bonferroni test). </p

Expressions of <i>Tmed2</i>, <i>Klc1</i>, <i>Nedd4, Hcrt</i>, <i>Meis2</i>, <i>Neurod6</i>, <i>Pitx2, Ptgds</i>, <i>Vtn</i>, Fn <i>1</i>, <i>Apod</i>, and <i>Igf2</i> genes in the hypothalamus of perinatal intact male and female mouse at embryonic day 16 (E16), E18, the day of birth (D0), and two days after the birth (D2).

<p>mRNA levels of these genes were determined semiquantitatively by RT-PCR followed by analysis with Image J from NIH. Gene expression levels in intact male (solid circle) and female hypothalamus (open circle) were indicated in relation to <i>Actb</i>. Values are means±SEM. Values marked with asterisks (* or **) are significantly different from female mice at each time point (P < 0.05 or P < 0.01, two-way ANOVA (sex and age as main factors) followed by the Bonferroni test). Section signs indicate significant effect between sex as a main factor of two-way ANOVA.</p

The number of Affymetrix probe ID, expressions of which were increased or decreased by neonatal estradiol bezoate (EB) treatment, in the hypothalamus of female mice.

<p>A, Numbers of genes showing a 2-fold or greater expression in EB-treated group compared with vehicle-treated control group; B, Numbers of genes showing a 0.5-fold or less expression in EB-treated group compared with vehicle-treated control group. Female mice were subcutaneously treated with EB (7.5 μg) on the day of birth and then the hypothalamus were collected at either 1, 3, 6, 12,or 24 h after the EB treatment. Difference of gene expression between EB- and vehicle-treated control mice was determined by GeneSpring GX 7.3 software. </p