Real-time Recombinase Polymerase Amplification Research Articles

Rapid and specific detection of Pentastiridius leporinus by recombinase polymerase amplification assay.

Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of an emerging and fast spreading sugar beet disease, the syndrome 'basses richesses' (SBR), in different European countries. The disease is caused by the γ-3-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the phytoplasma 'Candidatus Phytoplasma solani' which are exclusively transmitted by planthoppers and can lead to a significant loss of sugar content and yield. Monitoring of this insect vector is important for disease management. However, the morphological identification is time consuming and challenging as two additional cixiid species Reptalus quinquecostatus and Hyalesthes obsoletus with a very close morphology have been reported in sugar beet fields. Further, identification of females and nymphs of P. leporinus at species level based on taxonomic key is not possible. In this study, an isothermal nucleic acid amplification based on recombinase polymerase amplification (RPA) was developed to specifically detect P. leporinus. In addition, real-time RPA was developed to detect both adults (male and female) and nymph stages using pure or crude nucleic acid extracts. The sensitivity of the real-time RPA for detection of P. leporinus was comparable to real-time PCR, but a shorter time (< 7 min) was required. This is a first report for real-time RPA application for P. leporinus detection using crude nucleic acid templates which can be applied for fast and specific detection of this vector in the field.

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.

In this issue, we established rapid, cost-effective, and simple detection methods including recombines polymerase amplification with lateral flow dipstick (RPA-LFD) and real-time RPA for cyprinid herpesvirus 3(CyHV-3), and evaluated their sensitivity, specificity, and applicability, the real-time RPA method could achieve sensitive diagnosis of CyHV-3 within 1.3 copies per reaction, respectively. The real-time RPA method is 10-fold more sensitive than RPA-LFD method. The exact number of CyHV-3 can be calculated in each sample by real-time RPA. The sera from koi also can be tested in these methods. In addition, no cross-reaction was observed with other related pathogens, including carp oedema virus (CEV), spring viraemia of carp virus (SVCV), cyprinid herpesvirus 1(CyHV-1), cyprinid herpesvirus 2(CyHV-2), type I grass carp reovirus (GCRV-I), type II GCRV (GCRV-II), type III GCRV (GCRV-III), and Aeromonas hydrophila.

Phage based magnetic capture method as an aid for real time RPA detection of Salmonella spp. in milk

Salmonella is a major cause of foodborne diseases worldwide. Conventional rapid assays for detecting Salmonella in real samples often encounter severe matrix interference or detect the limited number of species of a genus, resulting the inaccuracy of detection. In this study, we developed a method that combined phage-based magnetic capture with real time recombinase polymerase amplification (RPA) for the rapid, highly sensitive, and specific detection of Salmonella in milk with an ultra-low detection limit. The Felix O-1 phage-conjugated magnetic beads (O-1 pMBs) synthesized in this method showed excellent capture ability for Salmonella spp. and ideal specificity for non-Salmonella strains. After O-1 pMBs-based magnetic separation, the limit of detection (LOD) of the real time RPA assay was 50 cfu/mL in milk samples, which was significantly increased by a magnitude of 3-4 orders. The method exhibited a high sensitivity (compatibility) of 100% (14/14) for all tested Salmonella serotype strains and an ideal specificity (exclusivity) of 100% (7/7) for the tested non-Salmonella strains. The entire detection process including Salmonella capture, DNA extraction, and real time RPA detection was completed within 1.5 h. Furthermore, milk samples spiked with 10 cfu/25 mL of Salmonella were detected positive after cultured in buffered peptone water for only 3 h. Therefore, the proposed method could be an alternative for the rapid and accurate detection of Salmonella.

Real-time MBDi-RPA using methyl-CpG binding protein 2: A real-time detection method for simple and rapid estimation of CpG methylation status

BackgroundAnalysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent. ResultsMBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging. Significance and noveltyReal-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.

Rapid and sensitive detection of large yellow croaker iridovirus by real-time RPA and RPA-LFD.

Large yellow croaker (Larimichthys crocea) is a vital marine-cultured species in China. Large yellow croaker iridovirus (LYCIV) can cause a high mortality rate in L. crocea. Rapid and convenient detection of LYCIV is an urgent demand for diagnosis. In this study, rapid and simple recombinase polymerase amplification (RPA), real-time RPA and RPA combined with lateral flow dipstick (RPA-LFD) methods were developed for the detection of LYCIV based on the conserved sequence of the LYCIV major capsid protein (MCP) gene. With these optimized RPA analyses, LYCIV detection could be completed within 20 min at 40°C. Both RPA and real-time RPA could detect viral DNA as low as 102 copies/μL, while the detection limit of RPA-LFD was 101 copies/μL, and there was no cross-reaction with other aquatic pathogens (KHV, CyHV-2, GCRV-JX01, SVCV, LCDV and LMBV). In practical evaluation of RPA, real-time RPA and RPA-LFD methods, the results showed consistency with the general PCR detection. In short, the developed RPA, real-time RPA and RPA-LFD analyses could be simple, rapid, sensitive and reliable methods for field diagnosis of LYCIV infection and have significant potential in the protection of LYCIV infection.

Development of nucleic acid extraction and real-time recombinase polymerase amplification (RPA) assay integrated microfluidic biosensor for multiplex detection of foodborne bacteria

Development of nucleic acid extraction and real-time recombinase polymerase amplification (RPA) assay integrated microfluidic biosensor for multiplex detection of foodborne bacteria

Rapid detection of Mycobacterium tuberculosis using recombinase polymerase amplification: A pilot study.

Tuberculosis remains one of the leading causes of death worldwide, especially in low- and middle-income countries. Tuberculosis treatment and control efforts are hindered by the difficulty in making the diagnosis, as currently available diagnostic tests are too slow, too expensive, or not sufficiently sensitive. Recombinase polymerase amplification (RPA) is a novel technique that allows for the amplification of DNA rapidly, at constant temperature, and with minimal expense. We calculated and compared the limit of detection, sensitivity, and specificity of two RPA-based assays for the diagnosis of pulmonary tuberculosis, using two sets of published primers. We also calculated and compared the assays' limits of detection and compared their performance using two different DNA extraction methods prior to amplification (a commercially available DNA extraction kit vs. the chelex method). The RPA-lateral flow assay had a limit of detection of 5 fg/μL of DNA, a sensitivity of 53.2%, and a specificity of 93.3%, while the real time-RPA assay had a limit of detection of 25 fg/μL of DNA, a sensitivity of 85.1%, and a specificity of 93.3%. There was no difference in assay performance when DNA extraction was carried out using the commercial kit vs. the chelex method. The real-time RPA assay has adequate sensitivity and specificity for the diagnosis of pulmonary tuberculosis and could be a viable diagnostic tool in resource-limited settings, but the lateral flow assay did not perform as well, perhaps due to the fact we used stored sputum specimens from a biorepository. More work is needed to optimize the RPA-lateral flow assay, to get a more accurate estimate of its specificity and sensitivity using prospectively collected specimens, and to develop both assays into point-of-care tests that can be easily deployed in the field.

The establishment of a recombinase polymerase amplification technique for the detection of mouse poxvirus

BackgroundEctromelia virus (ECTV) is the causative agent of mousepox in mice. In the past century, ECTV was a serious threat to laboratory mouse colonies worldwide. Recombinase polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method.ResultsIn this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of the crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed. The lowest detection limit of the ECTV RT- RPA assay was 100 copies of DNA mol-ecules per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 135 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement.ConclusionsIn conclusion, this RPA assay offers a novel alternative for the simple, sensitive, and specific identification of ECTV, especially in low-resource settings.

Rapid detection of Mucorales based on recombinase polymerase amplification and real-time PCR.

Mucormycosis, an invasive fungal disease with severe consequences, poses a significant threat to immunocompromised individuals. However, the timely and accurate identification of Mucorales infection continues to present difficulties. In this study, novel detection techniques utilizing recombinase polymerase amplification (RPA) and quantitative real-time polymerase chain reaction (qPCR) were developed, specifically targeting the mitochondrial rnl gene, in order to address this challenge. The specificity of the RPA and qPCR assay was assessed by adding genomic DNAs extracted from 14 non-targeted strains, as well as human and mouse blood. No false-positive results were observed. Additionally, genomic DNAs from 13 species in five genera of order Mucorales were tested and yielded positive results in both methods. To further evaluate the sensitivity of the assays, DNAs from Rhizopus oryzae, Mucor racemosus, Absidia glauca, Rhizomucor miehei, and Cunninghamella bertholletiae were utilized, with concentrations ranging from 1 ng/μL to 1 fg/μL. The limit of detection (LoD) for the RPA assay was determined to be 1 pg., with the exception of Rhizomucor miehei which had a LoD of 1 ng. The LoD for the qPCR assay varied between 10 fg and 1 pg., depending on the specific species being tested. Sensitivity analysis conducted on simulated clinical samples revealed that the LoD for RPA and qPCR assays were capable of detecting DNA extracted from 103 and 101 colony forming units (CFU) conidia in 200 μL of blood and serum, respectively. Consequently, the real-time RPA and qPCR assays developed in this study exhibited favorable sensitivity and specificity for the diagnosis of mucormycosis.

A Real-Time Recombinase Polymerase Amplification Assay for Specific Detection of Lumpy Skin Disease Virus.

Lumpy skin disease virus (LSDV) infection, accompanied by loss of hide quality, poor reproductive efficiency, consistent degenerative emaciation, and milk yield reduction of animals, causes severe economic implications in endemic zones. The heterologous attenuated goat pox (GTPV) vaccine (AV41 strain) was used in China to prevent LSDV infection. Only a few LSDV detection methods that distinguish LSDV from GTPV vaccine strains have been reported before. For simple, rapid, and specific detection of LSDV, the real-time recombinase polymerase amplification (RPA) method was established with the specific primers and probes designed according to the conserved regions of ORF132 gene sequences. The assay could be finished within 20 min at a constant temperature (39 °C). This method had a limit of detection (LOD) of 15 copies/μL for LSDV and no cross-reaction with the nucleic acids of goat pox virus, infectious bovine rhinotracheitis virus, Pasteurella multocida, and bovine healthy tissue. Furthermore, 43 clinical samples were detected by this method and the real-time PCR recommended by the World Organisation for Animal Health (WOAH), with a kappa value, was 0.94. These results demonstrated that the real-time RPA method for detecting LSDV developed in this study was characterized by high sensitivity and specificity, which has wide application value in the clinical diagnosis and detection of LSDV in China.

Recombinase polymerase amplification in combination with electrochemical readout for sensitive and specific detection of PIK3CA point mutations

As part of the ongoing evolution towards personalized anticancer therapy, mutation screening is becoming increasingly important and, therefore, also alternative detection strategies that allow for fast genetic diagnostics at the point of care. In the case of breast cancer, detecting cancer-associated point mutations in the PIK3CA gene is of particular importance for treatment decisions. We developed a recombinase polymerase amplification assay combined with an enzyme-linked electrochemical assay on multi-channel screen-printed gold sensors for specific and highly sensitive detection of three PIK3CA point mutations (H1047R, E545K, and E542K). Recombinase polymerase amplification (RPA) of the target sequences was optimized and characterized with a real-time RPA assay. Comparison with real-time PCR reveals that RPA is slightly inferior in terms of efficiency and sensitivity. However, the desired target DNA is successfully amplified at initial concentrations down to 100 copies μL−1. For electrochemical readout, biotinylated dCTP is used to label the target DNA during RPA. Single-stranded target DNA is produced with either asymmetric RPA or symmetric RPA followed by lambda exonuclease digestion. Characterization of the two different approaches in terms of sensitivity results in comparable detection limits (229 copies μL−1 and 224 copies μL−1, respectively), though RPA followed by lambda exonuclease digestion yields significantly higher currents. Finally, this method, together with a designed wild-type blocking oligo that inhibits binding of the wild-type target DNA during probe-target hybridization, allows for detecting the PIK3CA point mutations H1047R, E545K, and E542K in the presence of wild-type target DNA when the proportion of mutant target DNA is >20%.

Development and validation of real-time recombinase polymerase amplification-based assays for detecting HPV16 and HPV18 DNA.

Cervical cancer is the fourth leading cause of cancer-related death among women worldwide. Persistent human papillomavirus (HPV) infection is the principal cause of cervical cancer, with HPV16 and HPV18 accounting for about 70% of cases worldwide. Cervical screening is an effective secondary measure for preventing cervical cancer. Testing for HPV DNA is becoming increasingly important in cervical cancer screening. The existing PCR-based HPV detection technology has several disadvantages: the assays are time-consuming, and sophisticated equipment is required to control the temperature cycles. These drawbacks have led to the development of detection technologies based on isothermal amplification. Here, we present real-time recombinase polymerase amplification (RPA-exo)-based assays for single genotyping of either the E7 or the L1 segment of HPV16 or HPV18. These assays were highly sensitive, able to detect HPV in all clinical samples with Ct values below 34, and yielded results within 25 min. A dual-detection system capable of detecting both HPV16 and HPV18 in a single reaction was also developed based on the L1 gene. It has a limit of detection of approximately 10,000 copies of each genotype per reaction. The assays were validated with DNA extracted from 36 biopsy specimens and 42 exfoliated cell samples from 43 patients with cervical lesions at different stages. The RPA-exo system is a promising clinical detection platform with the advantages of yielding results rapidly and operating at a constant temperature, while being cost-effective and easy to use. IMPORTANCE HPV DNA screening is an effective approach for the prevention of cervical cancer. The novel real-time recombinase polymerase amplification-based HPV detection systems we developed constitute an improvement over the HPV detection methods currently used in clinical practice and should help to extend cervical cancer screening in the future, particularly in point-of-care test settings.

Rapid detection of Cryptosporidium spp. in diarrheic cattle feces by isothermal recombinase polymerase amplification assays

As a zoonotic parasite, Cryptosporidium spp. could cause severe diarrhea mainly in calves and children globally. Monitoring and prevention of Cryptosporidium spp.’s prevalence is of great significance in both economy and public health aspects. In this study, specific primers and probes were designed within the conserved region of 18S rRNA gene for Cryptosporidium spp. and recombinase polymerase amplification assays based on the fluorescence monitoring (real-time RPA) as well as combined with a lateral flow strip (LFS RPA) were developed. Both of the two RPA assays allowed the exponential amplification of the target fragment within 20 min. After incubation on a metal bath at 42 °C, the LFS RPA results were displayed on the lateral flow strip within 5 min while real-time RPA allowed the real-time observation of the results in Genie III at 39 °C. The RPA assays showed high specificity for Cryptosporidium spp. without any cross-reaction with other tested pathogens causing diarrhea in cattle. With the recombinant plasmid DNA containing the 18S rRNA gene of Cryptosporidium spp. serving as a template, the limit of detection for real-time RPA and LFS RPA assays were 14.6 and 12.7 copies/reaction, respectively. Moreover, the RPA assays were validated by testing diarrheic cattle fecal samples and compared with a real-time PCR. The positive ratio of Cryptosporidium spp. was 24.04 % (44/183) and 26.23 % (48/183) in both RPA assays and real-time PCR assay, respectively, and the kappa coefficient value was 0.942. The diagnostic specificity and diagnostic sensitivity of both RPA assays were 100 % and 91.67 %, respectively. Forty-one of 48 positive samples were successfully sequenced and four Cryptosporidium species were detected, including C. parvum (n = 20), C. andersoni (n = 17), C. bovis (n = 3) and C. ryanae (n = 1). The developed RPA assays are easy to operate and faster to obtain the detection results, and they are suiting for the point-of-care detection and facilitating the prevention and control of Cryptosporidium spp. infections.

Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 102 and 1.0 × 101 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

Evaluation of a real-time recombinase polymerase amplification assay for rapid detection of Schistosoma haematobium infection in a resource-limited setting

Evaluation of a real-time recombinase polymerase amplification assay for rapid detection of Schistosoma haematobium infection in a resource-limited setting

Real-time on-site detection of the three 'Candidatus Liberibacter' species associated with HLB disease: a rapid and validated method.

Huanglongbing (HLB) is a devastating disease that affects all commercial citrus species worldwide. The disease is associated with bacteria of three species of the genus 'Candidatus Liberibacter' transmitted by psyllid vectors. To date, HLB has no cure, so preventing its introduction into HLB-free areas is the best strategy to control its spread. For that, the use of accurate, sensitive, specific, and reliable detection methods is critical for good integrated management of this serious disease. This study presents a new real-time recombinase polymerase amplification (RPA) protocol able to detect the three 'Ca. Liberibacter' species associated with HLB in both plant and insect samples, validated according to European and Mediterranean Plant Protection Organization (EPPO) guidelines and tested on 365 samples from nine different geographic origins. This new protocol does not require nucleic acid purification or specialized equipment, making it ideal to be used under field conditions. It is based on specific primers and probe targeting a region of fusA gene, which shows a specificity of 94%-100%, both in silico and in vitro, for the 'Ca. Liberibacter' species associated with HLB. The analytical sensitivity of the new protocol is excellent, with a reliable detection limit in the order of 101 copies per microliter in HLB-infected plant and insect material. The repeatability and reproducibility of the new methods showed consistent results. Diagnostic parameters of the new RPA protocol were calculated and compared with the gold standard technique, a quantitative real-time PCR, in both crude extracts of citrus plants and insect vectors. The agreement between the two techniques was almost perfect according to the estimated Cohen's kappa index, with a diagnostic sensitivity and specificity of 83.89% and 100%, respectively, and a relative accuracy of 91.59%. Moreover, the results are obtained in less than 35min. All these results indicate the potential of this new RPA protocol to be implemented as a reliable on-site detection kit for HLB due to its simplicity, speed, and portability.

Rapid detection of EGFR mutation in CTCs based on a double spiral microfluidic chip and the real-time RPA method.

Circulating tumor cells (CTCs) are cells shed from primary or metastatic tumors and spread into the peripheral bloodstream. Mutation detection in CTCs can reveal vital genetic information about the tumors and can be used for "liquid biopsy" to indicate cancer treatment and targeted medication. However, current methods to measure the mutations in CTCs are based on PCR or DNA sequencing which are cumbersome and time-consuming and require sophisticated equipment. These largely limited their applications especially in areas with poor healthcare infrastructure. Here we report a simple, convenient, and rapid method for mutation detection in CTCs, including an example of a deletion at exon 19 (Del19) of the epidermal growth factor receptor (EGFR). CTCs in the peripheral blood of NSCLC patients were first sorted by a double spiral microfluidic chip with high sorting efficiency and purity. The sorted cells were then lysed by proteinase K, and the E19del mutation was detected via real-time recombinase polymerase amplification (RPA). Combining the advantages of microfluidic sorting and real-time RPA, an accurate mutation determination was realized within 2h without professional operation or complex data interpretation. The method detected as few as 3 cells and 1% target variants under a strongly interfering background, thus, indicating its great potential in the non-invasive diagnosis of E19del mutation for NSCLC patients. The method can be further extended by redesigning the primers and probes to detect other deletion mutations, insertion mutations, and fusion genes. It is expected to be a universal molecular diagnostic tool for real-time assessment of relevant mutations and precise adjustments in the care of oncology patients.

Establishment of a Real-Time Recombinase Polymerase Amplification for Rapid Detection of Pathogenic Yersinia enterocolitica

Yersinia enterocolitica is a zoonotic proto-microbe that is widespread throughout the world, causes self-limiting diseases in humans or animals and even leads to sepsis and death in patients with severe cases. In this study, a real-time recombinase polymerase amplification (RPA) assay for pathogenic Y. enterocolitica was established based on the ail gene. The results showed that the RPA detection for Y. enterocolitica could be completed within 20 min at an isothermal temperature of 38 °C by optimizing the conditions in the primers and Exo probe. Moreover, the sensitivity of the current RT-RPA was 10-4 ng/μL, and the study found that the assay was negative in the application of the genomic DNA of other pathogens. These suggest the establishment of a rapid and sensitive real-time RPA method for the detection of pathogenic Y. enterocolitica, which can provide new understandings for the early diagnosis of the pathogens.

Rapid detection of duck ingredient in adulterated foods by isothermal recombinase polymerase amplification assays

Duck is often used in meat fraud as a substitute for more expensive meats. Rapid detection of duck ingredient in meat products is of great significance for combating meat fraud and safeguarding the interests of consumers. Therefore, we aim to develop duck-specific recombinase polymerase amplification (RPA)-based assays for the rapid detection of duck ingredient in animal-derived foods. Using Cytb gene as target, the real-time RPA and RPA combined with lateral flow strips (LFS RPA) were developed successfully for the rapid detection of ducks in 20 min at 39 °C and 40 °C, respectively. The assays did not show cross-reactions with 6 other livestock and poultry. The developed RPA assays could detect 10 pg duck genomic DNA per reaction and 0.1 % (w/w) duck ingredient in duck and mutton mixed powder within 30 min, including a rapid nucleic acid extraction. Furthermore, duck ingredient could be detected in 30 different actual foods including heat-processed meats and blood products. Therefore, duck-specific real-time RPA and LFS RPA assays were successfully developed with good specificity and sensitivity, which could enable rapid detection of duck ingredient in the field and provide technical support for combating the meat fraud.

Specific biomarker mining and rapid detection of Burkholderia cepacia complex by recombinase polymerase amplification.

To mine specific proteins and their protein-coding genes as suitable molecular biomarkers for the Burkholderia cepacia Complex (BCC) bacteria detection based on mega analysis of microbial proteomic and genomic data comparisons and to develop a real-time recombinase polymerase amplification (rt-RPA) assay for rapid isothermal screening for pharmaceutical and personal care products. We constructed an automatic screening framework based on Python to compare the microbial proteomes of 78 BCC strains and 263 non-BCC strains to identify BCC-specific protein sequences. In addition, the specific protein-coding gene and its core DNA sequence were validated in silico with a self-built genome database containing 158 thousand bacteria. The appropriate methodology for BCC detection using rt-RPA was evaluated by 58 strains in pure culture and 33 batches of artificially contaminated pharmaceutical and personal care products. We identified the protein SecY and its protein-coding gene secY through the automatic comparison framework. The virtual evaluation of the conserved region of the secY gene showed more than 99.8% specificity from the genome database, and it can distinguish all known BCC species from other bacteria by phylogenetic analysis. Furthermore, the detection limit of the rt-RPA assay targeting the secY gene was 5.6 × 102 CFU of BCC bacteria in pure culture or 1.2 pg of BCC bacteria genomic DNA within 30 min. It was validated to detect <1 CFU/portion of BCC bacteria from artificially contaminated samples after a pre-enrichment process. The relative trueness and sensitivity of the rt-RPA assay were 100% in practice compared to the reference methods. The automatic comparison framework for molecular biomarker mining is straightforward, universal, applicable, and efficient. Based on recognizing the BCC-specific protein SecY and its gene, we successfully established the rt-RPA assay for rapid detection in pharmaceutical and personal care products.