A simple and rapid method for fish sex identification based on recombinase-aided amplification and its use in Cynoglossus semilaevis

Abstract

Fish sex identification is a basic technique of great importance for both fish genetic studies and fisheries. Due to the sexual reversal phenomenon in many fish species, a simple and rapid molecular identification method for fish genetic sex is urgently needed to suit versatile detection scenarios, such as point-of-need applications. In this study, we took Cynoglossus semilaevis as an example, established a recombinase-aided amplification (RAA)-based method for sex identification, and combined the RAA-detection with two result visualization approaches with distinct features, capillary electrophoresis (CE) and lateral flow dipstick (LFD). Specific primers and probe were designed to specifically detect the sex chromosome W of C. semilaevis in order to distinguish the genetic sex between males, pseudo-males and females. To evaluate the performance of our methods, the genetic sex for twenty-eight males, sixty-eight pseudo-males and fifty-four females were examined with the RAA-based method and classical PCR-based genotyping method, demonstrating the consistent results of sex identification between both methods. The RAA-LFD method is operationally simple, rapid (~ 30 min) and holds great potential for point-of-need applications of fish sex identification, including fishery fields. The method presented here could be effective for identifying fish gender with the ZW karyotype.

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests

“…LAMP, HDA, SDA, EXPAR, and CAMP are performed at high temperatures ranging from 60–65 °C [ 76 , 142 , 143 , 146 , 147 ]. RPA, RAA, MIRA, RCA, and NASBA have low amplification temperatures between 20–42 °C [ 55 , 68 , 103 , 148 ]. These amplification methods can also be sorted by total assay time.…”

“…Complementary strands are then generated from the ssDNA, where the single-stranded binding protein (SSB) binds to the ssDNA, and polymerase binds to the primer–DNA complex. Additionally, amplification occurs at a single temperature between 37–42 °C and within a short reaction time of 10–30 min [ 55 , 56 ]. This amplification method is advantageous over other isothermal amplification methods because of its simplicity, rapid amplification time, high sensitivity, and high specificity.…”

Development of a Rapid Sex Identification Method for Newborn Pigeons Using Recombinase Polymerase Amplification and a Lateral-Flow Dipstick on Farm

“…In the male samples, no signal was detected for any quantity, even on the 100 ng strip ( Figure 3 B). The sensitivity of the RPA-LFD for different DNA resources has varied, reported at values of 0.2 ng, 80 pg, 10 fg and 50 pg [ 28 , 30 , 31 , 32 ]. The RPA-LFD detector used in this study, displayed a higher sensitivity than many other similar techniques.…”

Development of real‐time recombinase polymerase amplification (RPA) and RPA combined with lateral flow dipstick (LFD) assays for the rapid and sensitive detection of cyprinid herpesvirus 3

“…Although polymerase chain reaction (PCR) and quantitative PCR (qPCR) are highly sensitive and specific, they need complex instruments, expensive reagents that need to be preserved at constant temperature and long in situ waiting time (Li et al, 2021). Loop-mediated isothermal amplification (LAMP) technology is an isothermal nucleic acid amplification technology developed in recent years (Notomi et al, 2020;Tomita et al, 2008). Under the action of best DNA polymerase, nucleic acid could be amplified by 10 9 -10 10 folds at 60-65°C for 15-60 min.…”