Development of a novel panel for blood identification based on blood-specific CpG-linked SNP markers.

Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.

A novel method for high-sensitivity detection of SARS-CoV-2 using dual double-quenched fluorescence probes.

SARS-CoV-2 has infected many people around the world, fast and accurate detection of the virus can help control the spread of the virus. RT-PCR is the gold standard method for SARS-CoV-2 detection. In this study, we improved the RT-PCR by proposing a novel method using dual double-quenched fluorescence probes. We used the improved probes to detect the plasmid DNA and RNA reference materials of SARS-CoV-2 respectively. When the improved probes were used, the background fluorescence intensity has reduced by 50%, the fluorescence increment has increased to 2.8 folds, and the Ct value has significantly reduced by 3 or more, indicating that the detection sensitivity has increased at least eight times. In addition, we also demonstrated that the improved probes have well performance in detecting SARS-CoV-2 with the minimum concentration of 6.2 copies/µL. This study will help biological companies develop better products for SARS-CoV-2 and other clinical pathogen infection.

Acute non-traffic traumatic spinal cord injury in the aging population: Analysis of the National Inpatient Sample 2005-2018.

BACKGROUND: This study aimed to determine risk factors for poor in-hospital outcomes in a large cohort of older adult patients with acute non-traffic traumatic spinal cord injury (tSCI). METHODS: This is a population-based, retrospective, observational study. Data of older adults ≥65 years with a primary discharge diagnosis of acute non-traffic tSCI were extracted from the US National Inpatient Sample (NIS) database 2005-2018. Traffic-related tSCI admissions or patients lacking complete data on age, sex and outcomes of interest were excluded. Univariate and multivariate logistic regression analysis was used to determine associations between variables and in-hospital outcomes. RESULTS: Data of 49,449 older patients (representing 246,939 persons in the US) were analyzed. The mean age was 79.9 years. Multivariable analyses revealed that severe International Classification of Disease (ICD)-based injury severity score (ICISS) (adjusted odds ratio [aOR] = 3.14, 95% confidence interval [CI]: 2.77-3.57), quadriplegia (aOR = 2.79, 95%CI: 2.34-3.32), paraplegia (aOR = 2.60, 95%CI:1.89-3.58), cervical injury with vertebral fracture (aOR = 2.19, 95%CI: 1.90-2.52), and severe liver disease (aOR = 2.33, 95%CI: 1.34-4.04) were all strong independent predictors of in-hospital mortality. In addition, malnutrition (aOR = 3.19, 95% CI: 2.93-3.48) was the strongest predictors of prolonged length of stay (LOS). CONCLUSIONS: Several critical factors for in-hospital mortality, unfavorable discharge, and prolonged LOS among US older adults with acute non-traffic tSCI were identified. In addition to the factors associated with initial severity, the presence of severe liver disease and malnutrition emerged as strong predictors of unfavorable outcomes, highlighting the need for special attention for these patient subgroups.

Maximum Spreading of Impacting Ferrofluid Droplets under the Effect of Nonuniform Magnetic Field.

This article investigates the maximum spreading of ferrofluid droplets impacting on a hydrophobic surface under nonuniform magnetic fields. A generalized model for scaling the maximum spreading is developed. It is observed that, if the magnetic field strength is zero, a ferrofluid droplet not only demonstrates similar spreading dynamics as the water droplet but also obeys the same scaling law for the maximum spreading factor. Therefore, this article emphasizes the effects of magnetic field strength. In this regard, a dimensionless parameter (Nm) is introduced as the ratio between inertial force and Kelvin force, with an assumption that the kinetic energy mainly transforms to thermal energy. This parameter allows us to rescale all experimental data on a single curve with the Padé approximant, which is applicable to a wide range of impact velocities and magnetic field strengths.

Typing of semen-containing mixtures using ARMS-based semen-specific CpG-InDel/STR markers.

Mixed traces are common biological materials found at crime scenes, and their identification remains a significant challenge in the field of forensic genetics. In recent years, DNA methylation has been considered as a promising approach for body fluid identification, and length polymorphic loci are still the preferred markers for personal identification. In this study, we used tissue-specific CpG sites with linked insertion or deletion (InDel) or short tandem repeat (STR) markers (CpG-InDel/STR) for both body fluid and individual identification. The tissue-specific CpG loci, which were all selected from the previous reports, were analyzed using a combination of bisulfite conversion and amplification refractory mutation system-multiprimer-PCR technology. InDels or STRs, which were selected within 400 bp upstream or downstream of the semen-specific CpG loci, were analyzed using a capillary electrophoresis platform. Eventually, we successfully constructed a panel containing 17 semen-specific CpG-InDel/STR compound markers compassing 21 InDels/STRs, 3 body-fluid positive controls (vaginal secretion-, saliva-, and blood-specific CpG), and 1 gender identification locus. Using this panel, full genotyping of individuals could be obtained successfully with 50 ng DNA input. Semen stains stored at room temperature for 7 months and degraded samples that were heat treated for up to 6 h were still identified efficiently. For semen containing mixed stains, it is also useful when the semen content is as low as 3.03%. Moreover, the cumulative discrimination power of this panel is 0.9999998. In conclusion, it is a robust panel enabling the validation of both the tissue source and individual identification of semen containing mixed stains and can be employed as an alternative solution for forensic case investigation.

Predicting human age by detecting DNA methylation status in hair.

Age prediction is of great importance for criminal investigation and judicial expertise. DNA methylation status is considered a promising method to infer tissue age by virtue of age-dependent changes on methylation sites. In recent years, forensic scientists have established various models to predict the chronological age of blood, saliva, and semen based on DNA methylation status. However, hair-inferred age has not been studied in the field of forensic science. In this study, we measured the methylation statuses of potential age-related CpG sites by using the multiplex methylation SNaPshot method. A total of 10 CpG sites from the LAG3, SCGN, ELOVL2, KLF14, C1orf132, SLC12A5, GRIA2, and PDE4C genes were found to be tightly associated with age in hair follicles. A correlation coefficient above 0.7 was found for four CpG sites (cg24724428 and Chr6:11044628 in ELOVL2, cg25148589 in GRIA2, and cg07547549 in SLC12A5). Among four age-prediction models, the multiple linear regression model consisting of 10 CpG sites provided the best-fitting results, with a median absolute deviation of 3.68 years. It is feasible to obtain both human identification and age information from a single scalp hair follicle. No significant differences in methylation degree were found between different sexes, hair types, or hair colors. In conclusion, we established a method to evaluate chronological age by assessing DNA methylation status in hair follicles.

DIP-microhaplotypes: new markers for detection of unbalanced DNA mixtures.

The identification of a suspect in a degraded and unbalanced DNA mixture has been a challenge for the standard short tandem repeat polymorphisms (STR) typing. Several methods have been introduced to solve this problem, such as DIP-STR, DIP-SNP, and SNP-STR markers. In this study, we proposed DIP-microhaplotype (deletion/insertion linked a chain of SNPs) as a kind of new genetic marker to type the unbalanced and degraded DNA mixture. We established the detection method with ten DIP-microhaplotype markers including 26 SNPs using allele-specific multiplex PCR followed by SNaPshot assay. This novel compound marker allows us to detect the minor DNA with a sensitivity of 1:100 to 1:1000 in a DNA mixture of any gender. Most of the DIP-microhaplotype markers had a relatively high probability of informative alleles with an average informative value (I value) of 0.308. In all, we proposed DIP-microhaplotype as a novel type of DNA marker for the detection of minor contributor from unbalanced DNA mixtures. Due to their inherent shorter length, higher polymorphism, and sensitivity, DIP-microhaplotypes are promising markers for the examination of the degraded and unbalanced mixtures in forensic stains or clinical chimeras.

Development of a multiplex methylation-sensitive restriction enzyme-based SNP typing system for deconvolution of semen-containing mixtures.

The identification of mixed stains has always been a difficult problem in personal identification in the forensic field. In recent years, tissue-specific methylation sites have proven to be very stable biomarkers for distinguishing tissue origin. However, it is still challenging to perform tissue source identification and individual identification simultaneously. In this study, we developed a method that uses tissue-specific methylation markers combined with single-nucleotide polymorphism (SNP) markers to detect semen from mixed biofluids and to identify individuals simultaneously. Semen-specific CpG markers were chosen from the literature and further validated utilizing methylation-sensitive restriction endonuclease (MSRE) combined with PCR technology. The neighboring SNP markers were searched in the flanking sequence of the target CpG within 400 bp, and SNP typing was then carried out through a single-base extension reaction followed by capillary electrophoresis. Eventually, a method of MSRE combined with SNaPshot that could detect 12 compound CpG-SNP markers was developed. Using this system, 10 ng of total DNA and DNA mixture with semen content up to 25% could be typed successfully. Moreover, the cumulative discrimination power of the system in the northern Chinese Han population is 0.9998. This study provides a valuable strategy for forensic practice to perform tissue origin and individual identification from mixed stains simultaneously.

A method of identifying the blood contributor in mixture stains through detecting blood-specific mRNA polymorphism.

In the past decades, messenger RNA (mRNA) biomarkers have been employed to identify the origin of body fluids in forensic medicine. We hypothesized that the polymorphism of mRNA could be applied to identify individuals in mixture samples composed of two body fluids. In this study, we selected five blood-specific mRNA biomarkers of venous blood (SPTB, CD3G, AMICA1, ANK1, and GYPA) that encompass 16 SNPs to identify the mixture contributor(s). Five specific gene markers for menstrual blood, semen, skin, saliva, and vaginal secretions were amplified and typed as body-fluid positive controls. We established the system of multiplex PCR and single base extension (SBE) reaction followed by CE. The amplicon size was between 90bp and 294bp. The peripheral blood specificity was examined against other human body fluids, including saliva, semen, skin, menstrual blood, and vaginal secretion. The 16 SNPs were peripheral blood specific and could be successfully typed in homemade mixtures which are composed of different body fluids with 1 ng peripheral blood mRNA added. This system showed a supersensitivity (1:100) in detecting the trace amount of peripheral blood mixed in other body fluids and a combined discrimination power (CDP) of 0.99929 in Chinese population. It was the first time to establish a method for identifying the blood donors and deconvoluting mixtures through detecting mRNA polymorphism with SNaPshot assay. This peripheral blood specific SNP typing system showed high sensitivity to the typing of blood source specific markers regardless of other body fluids in the mixture.

Circular RNAs as biomarkers and therapeutic targets in environmental chemical exposure-related diseases.

Chemical contamination in the environment is known to cause abnormal circular RNA (circRNA) expression through multiple exposure routes; yet, the underlying molecular mechanisms remain unclear. Non-coding RNAs (ncRNAs), especially circRNAs, play important roles in epigenetic regulation and disease pathogenesis; however, few studies have examined the function of circRNAs in chemical contamination-induced diseases. CircRNAs are covalently closed continuous loops that do not possess 5' and 3' ends, increasing their structural stability and limiting degradation by exoribonucleases. In addition, environmental chemical exposure-related diseases are often accompanied by aberrant expression of specific circRNAs and those circRNAs are often detected in tissues and body fluids. Based on these characteristics, circRNAs may serve as candidate biomarkers for the diagnosis of diseases related to environmental chemical exposure. Here, we review the generation and function of circRNAs, and the possible molecular mechanisms underlying the regulation of environmental chemical exposure-related disorders by circRNAs. This is the first comprehensive review of the relationship between environmental chemical exposure and circRNAs in chemical exposure-induced diseases.

A new set of DIP-SNP markers for detection of unbalanced and degraded DNA mixtures.

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP-SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP-SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP-SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP-SNPs in a Chinese Han population. The DIP-SNPs were capable of detecting the minor contributor's allele in home-made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1 -10 ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP-SNPs may serve as a valuable tool in detection of UDM in forensic medicine.

A set of 14 DIP-SNP markers to detect unbalanced DNA mixtures.

Unbalanced DNA mixture is still a difficult problem for forensic practice. DIP-STRs are useful markers for detection of minor DNA but they are not widespread in the human genome and having long amplicons. In this study, we proposed a novel type of genetic marker, termed DIP-SNP. DIP-SNP refers to the combination of INDEL and SNP in less than 300bp length of human genome. The multiplex PCR and SNaPshot assay were established for 14 DIP-SNP markers in a Chinese Han population from Shanxi, China. This novel compound marker allows detection of the minor DNA contributor with sensitivity from 1:50 to 1:1000 in a DNA mixture of any gender with 1 ng-10 ng DNA template. Most of the DIP-SNP markers had a relatively high probability of informative alleles with an average I value of 0.33. In all, we proposed DIP-SNP as a novel kind of genetic marker for detection of minor contributor from unbalanced DNA mixture and established the detection method by associating the multiplex PCR and SNaPshot assay. DIP-SNP polymorphisms are promising markers for forensic or clinical mixture examination because they are shorter, widespread and higher sensitive.

A SNaPshot assay for detection of 45 mutations in the SCN5A gene in the Chinese Han Population.

Sudden cardiac death (SCD) occurs frequently in forensic practice and results in no visible pathological changes that can be detected in an autopsy. In recent years, the genetic background has been emphasized when examining SCD cases. The aim of this study is to establish a feasible system to detect SCD-related genes for forensic DNA laboratories. Forty-five reported SCD-associated SNPs from sodium voltage-gated channel alpha subunit 5 (SCN5A) were considered in our experiment. We established a SNaPshot assay for the typing of 45 SNPs using multiplex PCR and the minisequencing technique. Two multiplex PCRs were performed and optimized to cover 14 and 16 DNA fragments. The SCD victims came from the Chinese Han population residing in Shanxi and Chongqing provinces and were examined and compared with a non-SCD group and with normal healthy individuals. A missense mutation at rs1805124 (H558R) was detected in the Chinese Han population in this study. A SNaPshot assay can be performed in any forensic DNA laboratory and would be capable of meeting the increasing demand for SCD detection. This method would also be beneficial for screening at-risk in family members of SCD victims.

[The mechanism of histone lysine methylation of plant involved in gene expression and regulation].

Histone modification is one important sort of the epigenetic modifications, including acetylation, formylation, methylation, phosphorylation, ubiquitination and SUMOylation. By forming a complicated network, these modifications control the expression of genes. Histone methylation occurs mainly on the lysine residues, and plays a key role during flowering and stress response of plants, through changing the methylation status of lysine residues and the ratio of methylation. Triple-methylation of H3K4 promotes FLC expression but triple-methylation of H3K27 inhibits its expression. H3K4me3 activates the expression of PtdIns5P gene to initiate lipid synthesis signal pathway in response to drought stress. On the contrary, the low levels of H3K27me3 induce the expression of COR15A and ATGOLS3, which encode for low temperature protective proteins of chloroplast (Cor15am) and Galactional Synthase (GOLS), in order to resist cold stress. In this review, we summarize the molecular mechanisms of histone lysine methylation involved in DNA methylation, plant flowering and stress response.

Effects of foot reflexology massage on pregnant women: a systematic review and meta-analysis of randomized controlled studies.

To explore the effects of foot reflexology massage on anxiety, pain, duration of labor, labor satisfaction, blood pressure, pulse rate and respiratory rate in pregnant women. We systematically searched eight databases for randomized controlled studies on the effects of foot reflexology massage on pregnant women. The inclusion criteria were as follow: participants were pregnant woman; the intervention is foot reflexology or foot massage; the control intervention is placebo, usual care, or no intervention; outcome indicators included pain, anxiety, birth satisfaction, duration of labor, blood pressure, pulse, and respiration; and study type was randomized controlled study. Studies that did not meet the above requirements were excluded. We assessed the quality of the included studies using the Physiotherapy Evidence Database scale, the risk of bias using the Risk of Bias 2.0 tool, and the level of evidence for the outcomes using the Grading of Recommendations Assessment Development and Evaluation. We used Review Manager 5.3 for data analysis and generated funnel plots to assess publication bias. In addition, sensitivity analysis was used to test the stability of the results. A total of 13 randomized controlled studies with 1189 participants were included in this study. Compared to the control group, foot reflexology massage reduced anxiety and pain in pregnant women, shortened the three stages of labor, and increased birth satisfaction. In addition, it also reduced the pulse rate and respiratory rate of pregnant women, but not for blood pressure. Foot reflexology massage can significantly reduce anxiety and pain, shorten the duration of labor, increase birth satisfaction, and stabilize vital signs in pregnant women. It is a safe and non-invasive form of complementary therapy.PROSPERO registered number: CRD42022359641. URL: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=359641 .

Identification of the body fluid donor in mixtures through target mRNA cSNP sequencing.

In forensic practice, mixture stains containing various body fluids are common, presenting challenges for interpretation, particularly in multi-contributor mixtures. Traditional STR profiles face difficulties in such scenarios. Over recent years, RNA has emerged as a promising biomarker for body fluid identification, and mRNA polymorphism has shown excellent performance in identifying body fluid donors in previous studies. In this study, a massively parallel sequencing assay was developed, encompassing 202 coding region SNPs (cSNPs) from 45 body fluid/tissue-specific genes to identify both body fluid/tissue origin and the respective donors, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin. The specificity was evaluated by examining the single-source body fluids/tissue and revealed that the same body fluid exhibited similar expression profiles and the tissue origin could be identified. For laboratory-generated mixtures containing 2-6 different components and mock case mixtures, the donor of each component could be successfully identified, except for the skin donor. The discriminatory power for all body fluids ranged from 0.997176329 (menstrual blood) to 0.99999999827 (blood). The concordance of DNA typing and mRNA typing for the cSNPs in this system was also validated. This cSNP typing system exhibits excellent performance in mixture deconvolution.

[Expression regulation of plant ascorbate peroxidase and its tolerance to abiotic stresses].

Ascorbate peroxidase (APX), a type I heme peroxidase, catalyzes oxidation of ascorbic acid. It possesses a high degree of specificity to ascorbic acid. APX gene cluster consists of four sub-clusters: the gene clusters of cytosol, chloroplast, mitochondria, and peroxidase. As a key component of hydrogen peroxide detoxification system, the ascorbate-glutathione cycle, APX plays a vital role in the metabolism of H2O2 of plant cells. Studies showed that APX is one of the most important enzymes, which modulate the cellular H2O2 level in redox signaling system. The expression mechanisms of APX isoenzymes are quite complex. Briefly, cytosolic APX is regulated by a variety of signals; two chloroplastic APX isoenzymes are tissue-dependently regulated by alternative splicing. Generated APXs could regulate redox signaling in cells, which further boosts plants tolerance to abiotic stresses. This review focuses on recent advances concerning catalytic prop-erties, physiological function, and gene expressing regulation and abio-stress responding mechanism of APX.

Thallium(III) exposure alters diversity and co-occurrence networks of bacterial and fungal communities and intestinal immune response along the digestive tract in mice.

The gut microbiota, which includes fungi and bacteria, plays an important role in maintaining gut health. Our previous studies have shown that monovalent thallium [Tl(I)] exposure is associated with disturbances in intestinal flora. However, research on acute Tl(III) poisoning through drinking water and the related changes in the gut microbiota is insufficient. In this study, we showed that Tl(III) exposure (10 ppm for 2 weeks) reduced the alpha diversity of bacteria in the ileum, colon, and feces of mice, as well as the alpha diversity of fecal fungi. In addition, principal coordinate analysis showed that Tl(III) exposure had little effect on the bacterial and fungal beta diversity. LEfSe analyses revealed that Tl(III) exposure altered the abundance of intestinal bacteria in the digestive tract and feces. Moreover, Tl(III) exposure had little effect on fungal abundance in the ileum, cecum, and colon, but had a considerable effect on fungal abundance in feces. After Tl(III) exposure, the fungal composition was more disrupted in feces than in the intestinal tract, suggesting that feces can serve as a representative of the gut mycobiota in Tl(III) exposure studies. Intra-kingdom network analyses showed that Tl(III) exposure affected the complexity of bacterial-bacterial and fungal-fungal co-occurrence networks along the digestive tract. The bacterial-fungal interkingdom co-occurrence networks exhibited increased complexity after Tl(III) exposure, except for those in the colon. Additionally, Tl(III) exposure altered the intestinal immune response. These results reveal the perturbation in gut bacterial and fungal diversity, abundance, and co-occurrence network complexity, as well as the gut immune response, caused by Tl(III) exposure.

BézierSeg: Parametric Shape Representation for Fast Object Segmentation in Medical Images.

BACKGROUND: Delineating the lesion area is an important task in image-based diagnosis. Pixel-wise classification is a popular approach to segmenting the region of interest. However, at fuzzy boundaries, such methods usually result in glitches, discontinuity or disconnection, inconsistent with the fact that lesions are solid and smooth. METHODS: To overcome these problems and to provide an efficient, accurate, robust and concise solution that simplifies the whole segmentation pipeline in AI-assisted applications, we propose the BézierSeg model which outputs Bézier curves encompassing the region of interest. RESULTS: Directly modeling the contour with analytic equations ensures that the segmentation is connected and continuous, and that the boundary is smooth. In addition, it offers sub-pixel accuracy. Without loss of precision, the Bézier contour can be resampled and overlaid with images of any resolution. Moreover, clinicians can conveniently adjust the curve's control points to refine the result. CONCLUSIONS: Our experiments show that the proposed method runs in real time and achieves accuracy competitive with pixel-wise segmentation models.

A recombinase polymerase amplification (RPA) combined with strip visualization method for RNA-based presumptive tests of saliva and vaginal secretion.

Identifying the origin of body fluids is a critical step in a forensic investigation. One widely used method to identify human body fluids is based on the color visualization of immune antigen detection strips for detecting hemoglobin in blood and prostate-specific antigen in semen. It is highly imperative to construct an easy-to-perform, mRNA-based method for the point-of-care identification of other human body fluids, such as saliva and vaginal secretion. Here, we established specific strips with the mRNA markers STATH (for saliva) and SPINK5 (for vaginal secretion) via reverse transcription recombinase polymerase amplification (RT-RPA) and lateral flow dipstick (LFD) assays (RT-RPA-LFD). RT-RPA could be accomplished in a single tube at a wide temperature range of 30-42 â„ƒ within 10-25 min if we do not count time for RNA extraction. The diluted RPA products were added onto the LFD strip pad to visually observe the color change of the Control/Test line. The tissue specificity and detection limit of the assays were evaluated using the optimized reaction conditions of RPA at 37 â„ƒ for 15 min. The positive signals of STATH were observed both in saliva and nasal secretions. SPINK5 was positive in a template-dependent manner in 4 out of 30 female urine samples in addition to vaginal secretion and menstrual blood samples. Cross-reactions were not detected in semen, skin swabs, sweat, or male urine. Both assays were capable of detecting aged samples, which were stored for 180 days (saliva) or 300 days (vaginal secretion) at room temperature. Moreover, saliva or vaginal secretion was successfully detected in all kinds of mixtures made from various body fluids. Overall, the rapid strip test method by the RT-RPA-LFD assay is simple, time-saving and highly sensitive for estimating the tissue origin of saliva and vaginal secretion. This method for the rapid RNA-based presumptive tests of the tissue type of body fluids is easy to perform prior to a multiplex mRNA analysis, which can demonstrate more reliable saliva or vaginal secretion identification.