Recombinase polymerase amplification with polymer flocculation sedimentation for rapid detection of Staphylococcus aureus in food samples.

Currently, rapid, sensitive, and convenient visual detection methods for Staphylococcus aureus (S. aureus) are scarce. In this study, a novel detection method based on recombinase polymerase amplification (RPA) and polymer flocculation sedimentation (PFS) was developed. Twelve effective primer combinations derived from four forward primers F1, F2, F3, F4, and three reverse primers R1, R2, R3 targeting the nuc gene of S. aureus were designed and screened by a polymerase chain reaction and RPA methods. RPA reaction conditions, including temperature, time, and volume as well as PEG8000 and NaCl concentrations range, were optimized. Moreover, the specificity and sensitivity of the RPA-PFS assay were further analyzed. Finally, the potential use of the RPA-PFS assay was evaluated using artificially S. aureus contaminated food samples, including pork, beef, shrimp, fish, cheese, cabbage, leftover rice, egg, milk, and orange juice. Results showed that the SA5 (F2/R2) combination was the optimal primer candidate. The optimal temperature range, the shortest time and the minimal volume of RPA reaction were 40-42 °C, 10 min and 10 µL, respectively and the optimal PEG8000/NaCl concentrations were 0.2 g/mL and 2.5 M, respectively, for the adsorption between magnetic beads and RPA products. The RPA-PFS method could detect as little as 13 fg genomic DNA of S. aureus and was also specific for five target S. aureus as well as twenty-seven non-target foodborne bacteria. The limit of detection of RPA-PFS for S. aureus in artificially contaminated food samples was 38 CFU/mL (g). Besides, RPA-PFS has directly been judged by the naked eye and has totally taken less than 20 min. In short, the assay RPA-PFS developed in this study is a rapid, sensitive, and specific visual detection method for S. aureus.

Sensitive and rapid visual detection of Salmonella Typhimurium in milk based on recombinase polymerase amplification with lateral flow dipsticks.

Salmonella Typhimurium (S. Typhimurium) can cause serious foodborne diseases. In this study, an assay combining recombinase polymerase amplification (RPA) with lateral flow dipsticks (LFD) was developed to detect S. Typhimurium in milk. The RPA forward primers STF1, STF2, STF3, the reverse primer STR labeled with digoxin, and the probe STProb labeled with FAM were designed and screened to produce RPA products for LFD detection. The RPA reaction volume, temperature, and time were then optimized, and the sensitivity and specificity of the developed method were analyzed. Finally, the RPA-LFD method was evaluated using milk artificially contaminated with S. Typhimurium. Results indicated that the primer pair STF1/STR is the optimal combination for detecting the bacterium. The minimum volume, shortest time, and optimal temperature of the RPA reaction were 10 µL, 10 min, and 40-42 °C, respectively. The limit of detection of RPA-LFD for detecting the genomic DNA of S. Typhimurium was 1 fg, which is 5 and 10 times lower than the corresponding limits of RPA-agarose gel electrophoresis (AGE) and PCR-AGE, respectively. Testing with 29 other foodborne bacteria as controls revealed that RPA-LFD was highly specific for S. Typhimurium. RPA-LFD can detect S. Typhimurium at concentrations as low as 1.95 CFU/mL in artificially inoculated milk samples and is thus 10 times more sensitive than PCR. Hence, the RPA-LFD assay established in this study could be a potential point-of-care/need test for S. Typhimurium, especially in areas with limited resources.

Development of duplex PCR-ELISA for simultaneous detection of Salmonella spp. and Escherichia coli O157: H7 in food.

In the current study, a duplex PCR-ELISA method was developed targeting the specific genes, invA of Salmonella spp. and rfbE of Escherichia coli O157: H7, to detect one or both bacteria in food. In brief, PCR product amplified by PCR primer labeled with digoxin at the 5'-end and a probe labeled with biotin at the 3'-end can form dimer by nucleic acid hybridization which can be captured by binding of biotin to streptomycin coated in ELISA plate before using enzyme-labeled anti-digoxin antibody and substrate to develop color. Also, evaluation of the duplex PCR-ELISA method was conducted in different food samples including milk, juice, cabbage, shrimp, chicken, pork and beef. Results indicated that the duplex PCR-ELISA developed here was specific when using 25 non-target bacteria strains as controls and was sensitive with a limit of detection (LOD) of 1 CFU/mL, 1, 000 times higher than that of the duplex PCR method and was repeatable regardless of inter- and intra-batch variations. The duplex PCR-ELISA method established in the present study has proven to be highly specific, sensitive and repeatable. It has the potential to be applied in such fields as clinical diagnosis of food-borne diseases, food hygiene monitoring and pathogen detection in food.

[RAPD and SCAR molecular markers linked to the sexuality of cycads (Cycas tanqingii D. Y. Wang)].

The random amplified polymorphic DNA (RAPD) technique was used to amplify DNA fragment, aiming at finding markers linked to the sex trait in Cycas tanqingii D. Y. Wang. A total number of 160 random primers were screened in the RAPD-PCR and more than 2500 RAPD fragments were generated from the male or the female plants. One fragment of about 500 bp was amplified steadily and repeatedly by the S0465 (CCCCGGTAAC) primer only from female plants but not male plants. The RAPD marker was then converted into female-linked dominant SCAR (Sequence Characterized Amplified Regions) marker named STQC-S465-483. The development of this sex-linked SCAR marker provides a possibility of identifying the sex of Cycas tanqingii before sexual maturation, which is very important to in situ or ex situ conservation.

[Determination of SARS-CoV by simplified nested fluorescent RT-PCR].

OBJECTIVE: To establish a simple rapid and sensitive nested RT-PCR method for detection of SARS coronavirus RNA by designing the specific primers for SARS and optimizing the parameters for PCR. METHODS: Primers and fluorescent probes were designed according to the sequences of SARS coronavirus genes available from GenBank. The optimization of the parameters for PCR was performed in PE 7700 thermal cycle. The 36 serum samples and 40 mouthwash of SARS patients and 80 samples of healthy people were tested. RESULTS: The positive rate of patient serum and mouthwash was 33.6%, (12/36) and 67.5%, (27/40), respectively, while the positive rate of healthy people was zero (0/160). CONCLUSION: The simple nested RT-PCR method was a rapid, efficient and sensitive one for SARS early diagnosis.

[Assessment of genetic diversity of Rehmannia glutinosa germplasm detected by RAPDs and ISSRs].

RAPD and ISSR markers were used to assess the germplasm genetic diversity among 10 individuals of Rehmannia glutinosa, including 8 cultivars and 2 virus-free lines micropropagated by tip tissue culture. 17 RAPD primers and 10 ISSR primers, with polymorphic and informative patterns, were selected from a total of 80 RAPD ones and 44 ISSR ones to determine these individuals' genetic diversity. The 17RAPD primers and 10 ISSR primers generated 177RAPDfragments and 110 fragments, respectively. The number of effective loci, the percentage of polymorphic loci, Shannon's Information index (I) and effective number of alleles (Ne) is in turn109, 61.58%, 0.3135, 1.3641 for RAPD makers, and 79, 71.82 %, 0.3577 and 1.4037 for ISSR markers; Jaccard's genetic similarity matrice and dendrograms for the 10 individuals were formed based on RAPD and ISSR-generated polymorphic bands. In dendrograms, they could be divided into two groups: one group containing six individuals such as Zupei 85.5, Datian 85.5, jinzhuangyuan, Jinbai, Zupei 9302 and Datian9302; the other composed of 4 ones such as Beijing No.1, Dahongpao, Dihuang 9104 and wild dihuang; the correlation coefficient of 0.649 between RAPD and ISSR markers GSs indicated that these two markers were significantly correlated. The results revealed that RAPD and ISSR markers were suitable for assessment of germplasm genetic diversity of Rehmannia glutinosa, and ISSR marker was superior to RAPD marker.