The Shigella strain used in this study was isolated from monkey by Kunming Biomed International. Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, and Pseudomonas aeruginosa were used for specificity testing as shown in Supplementary Table S1.

Shigella was used to produce antibodies in BALB/c mice. Prior to immunization, a blood sample from each mouse was collected. The blood was incubated for 1 h at 37°C and was then centrifuged at 3000 rpm for 20 min at 4°C. The serum was gathered as a negative control. Mice were first immunized by back multipoint subcutaneous injection using a mixture of 100 μL (10⁸ inactivated bacteria) of diluted Shigella with an equal volume of Freund’s complete adjuvant. After an interval of 2 weeks, injections were given with a mixture of 100 μL of diluted Shigella in incomplete Freund’s adjuvant. Subsequently, the blood was obtained 1 week after the last injection, the serum was collected as described above, and polyclonal antibody specific for Shigella was produced. Animal experiments and procedures were performed ethically according to the Guidelines for Animal Experiments and approved by the Committee on Experimental Animals at Kunming University of Science and Technology.

Indirect enzyme-linked immunosorbent assay (ELISA) was used to determine the titer of the serum. The cultured Shigella (inactivated) was diluted to a concentration of 10⁸ CFU/mL in phosphate-buffered saline (PBS; 2.7 mmol/L KCl, 137 mmol/L NaCl, 2 mmol/L KH₂PO_4, 10 mmol/L Na₂HPO_4, pH 7.4) and then was coated on 96-well plates at 100 μL and incubated for 3 h at 37°C. The coated wells were washed three times with PBS containing 0.05% Tween-20 and then blocked with 100 μL of blocking buffer (5% skimmed milk in PBS-T) for 2 h at 37°C. Followed by three washes with PBS-T buffer, the coated wells were incubated with 100 μL of serum against Shigella with different dilutions (from 1:200 to 1: 409600) at 37°C for 2 h. After incubation, the coated wells were washed three times with PBS-T buffer and assayed using the goat anti-mouse IgG (H+L) HRP (GenScript, United States) as a secondary antibody. The optical density of each well was then measured at 450 nm using a microplate reader (Bio-Rad) (Tang et al., 2016).

To ensure that the hyperimmune serum of Shigella could recognize and capture the bacterium, a bacteriostatic experiment was performed. The cells were cultured overnight and then were diluted to concentrations of 10⁸, 10⁶, and 10⁴ CFU/mL with sterile water. Then, 50 μL of bacterial liquid culture was applied to LB solid medium (0.5% Yeast Extract, 1% Tryptone, 1.5% Agar, 1% NaCl, pH 7.0) as a blank control. Separately, 50 μL bacteria culture plus 2 μL multiple antiserum or 50 μL bacterial culture with 2 μL negative serum (from mice that were not immunized with Shigella) were also applied to LB solid medium as positive and negative controls, respectively. The agar plates were cultured at 37°C for 12 h. After culturing, the inhibitory effect of anti-Shigella serum on the growth of different concentrations of Shigella was measured.

The genomic DNA of all bacteria used in this study was extracted by a Bacteria Genomic DNA kit (Zomanbio, China). The DNA concentration was measured using an ultraviolet spectrophotometer and stored at -20°C before use.

The outer primers of the LAMP assay (F3: 5′-ATACCGTCTCTGCACGCA-3′ and B3: 5′-GCCTTCTGATGCCTGATGG-3′) were used to perform PCR reaction in a 25 μL volume which contained 12.5 μL of 2 × TSINGKE Master Mix (containing DNA polymerase 1 U, 1.5 mM MgCl₂, 200 mM each dNTP), 10 μM of each primer, and 50 ng genomic DNA. The reactions were carried out in GeneAmp PCR System 9700 with the following amplification conditions: pre-denaturing at 95°C for 5 min, followed by 34 cycles of denaturing at 95°C for 30 s, annealing at 57°C for 30 s, and extending at 72°C for 30 s, and a final extension at 72°C extension for 7 min. A total of 5 μL of the PCR product was analyzed by electrophoresis in 1% agarose gel.

The LAMP assay was performed as described by Notomi et al. (2000) and Savan et al. (2004). LAMP requires a set of four primers (B3: 5′-GCCTTCTGATGCCTGATGG-3′, F3:5′-ATACCGTCTCTGCACGCA-3′, BIP:5′-CTTTCGCTGTTGCTGCTGATGCTTTTCCGGAGATTGTTCCATGTGA-3′, and FIP:5′-CTGTCGAAGCTCCGCAGAGGTTTTGGATTC CGTGAACAGGTCG-3′), recognizing six distinct target sites of the DNA sequence. Primers used for the LAMP reaction were designed using the primer design software Primer Explorer V5, primer software available on the Net laboratory website¹, and targeted the conserved C-terminal region of the ipaH gene. Primers FIP and BIP were composed of the complementary sequence of the F1, B2, and the direct sequence of F2, B1, respectively. Primers F3 and B3 were located outside the F2 and B2 regions, respectively. The LAMP reaction was performed in a total volume of 25 μL of a mixture containing 1 μL DNA template, 20 pM each of the FIP and BIP primers, 2.5 pM each of the F3 and B3 primers, 1 μL of Bst DNA polymerase (Eiken Chemical Co., Ltd., Tokyo, Japan), 2.5 μL of 10 × LAMP buffer (200 mmol/L Tris–HCl, 100 mmol/L (NH₄)₂SO₄, 100 mmol/L KCl, 1% Triton X-100, pH 8.8), 150 nM Mg²⁺, 40 nM dNTP mixture, and 9.5 μL nuclease-free water. The reaction of the mixture was initiated by an incubation of 63°C for 50 min and terminated at 80°C for 2 min. A total of 5 μL of the product was analyzed by separation on 3% agarose gel.

Protein A/G-coated magnetic beads (Biotool, United States) were suspended, and transferred from 25 μL to a 1.5 mL tube. The magnetic beads were washed extensively with binding buffer (150 mmol/L NaCl, 50 mmol/L Tris, 0.5% Tween-20, pH 7.5). Then, magnetic separation was performed, the supernatant was removed and 200 μL of fresh binding buffer was added. The mouse anti-Shigella serum was diluted (1:4,000) into 5% nonfat dry milk in PBS containing 0.05% Tween-20. A total of 50 μL of mouse anti-Shigella serum was transferred into the 1.5 mL tubes and incubated for 2 h at 37°C. Magnetic separation was performed again. The nonbinding anti-Shigella antibody in tubes was then removed and the beads were washed three times by PBS-T.

After preparation of immunomagnetic beads, the immunomagnetic beads were used to capture Shigella at 37°C for 0.5 h. Then, 10 μL solution A (125 mM NaOH, 1 mM EDTA, 0.1% Tween-20) was added to the tubes, followed by incubation at 65°C for 10 min to lyse the bacteria cells. Finally, 10 μL solution B (125 mM HCl, 10 mM Tris) was added to terminate the reaction. Then, 1–6 μL of bacteria lysate was used as template for LAMP amplification as described above.

In order to test the specificity of the designed primers, we amplified the genomic DNA of five common food-borne pathogenic bacteria collected in this study by using PCR, LAMP, and IC-LAMP.

The constructed templates were used to detect the sensitivity of the PCR, LAMP, and IC-LAMP. The plasmid concentration was calculated using the deduced polynomial model described as:

In order to test the applicability of IC-LAMP technology in milk, Shigella was mixed with pasteurized milk samples, purchased from a grocery store in Kunming and identified as Shigella-negative by traditional culture techniques and PCR method. First, in order to check the minimal detectable colony forming units (CFUs), the cultures of Shigella strains were serially diluted (10^-1–10^-9). The CFUs of the plate were counted after 24 h at 37°C. Next, the aliquots of 100 μL appropriate dilutions with Shigella were inoculated into the milk samples, for final concentrations of Shigella of approximately 8.7 × 10⁶, 8.7 × 10⁵, 8.7 × 10⁴, 8.7 × 10³, 8.7 × 10², 8.7 × 10¹, and 8.7 × 10⁰ CFU/mL. The milk samples were transferred to a tube, which containing magnetic beads, antibody, and incubated at 37°C for 30 min. After enrichment of the cells, the tubes were washed three times with PBS-T, followed by the addition of 10 μL of solution A and incubation at 65°C for 10 min. Finally, 10 μL of solution B was added to terminate the reaction. Then, 1–6 μL of bacteria lysate was used for IC-LAMP. A non-contaminated milk sample was used as a negative control. Each milk sample was tested five times with the IC-LAMP.

Shigella screening is a necessary component of inspection for laboratory monkey export. We tested direct detection of Shigella in monkey clinical samples from feces and/or anal swabs. To do this, 46 clinical samples were collected from monkeys for IC-LAMP assay detection and analysis. These samples were first suspended in 1 mL sterile LB liquid medium and incubated at room temperature for 1 h, before the suspension was transferred to 1.5 mL tubes that contained magnetic beads and antibody. All tubes were reacted at 37°C for 0.5 h. After enrichment of the cells, the tubes were washed three times with PBS-T, and then 10 μL of solution A was added and the mixture was incubated at 65°C for 10 min. Next, 10 μL of solution B was added to terminate the reaction. After this treatment, 1–6 μL of bacteria lysate was used for IC-LAMP. A non-contaminated clinical sample was used as a negative control.

In the animal room experimental area, a cotton swab was used to take a small amount of fresh manure. The middle of the stool was used for the sample to avoid urine, food residue, or other contaminants. As an alternative, anal swabs were taken from primates if fresh feces were not available.

The swabs or anal swabs with sticky feces were seeded on a petri dish using a zoning method. After inoculation, the dish was inverted (agar plate bottom up), numbered, and cultured at 37°C for 18–24 h. Care was taken to avoid scratching the surface of the medium when applying the samples.

There were colorless, round, flat, or slightly raised colonies that were smooth and moist, with neat edges, and about 2 mm in diameter. Shigella colonies are generally larger, translucent, and sometimes may appear flat, with an irregular edge of a rough type of colony.

In the biological safety cabinet, an inoculation ring was used to pick 2–3 suspicious colonies that were then cultured on a Triple Sugar Iron Agar Medium at 37°C for 18–24 h. If the selected colonies were Shigella, the slope does not produce acid, so the bottom of the acid does not produce gas, and the hydrogen sulfide is negative.

Onto a clean glass slide, a small amount of Shigella multivalence diagnostic serum (Lanzhou Institute of Biological Products Co., Ltd, China) was applied. The inoculation ring was then used to take Salmonella–Shigella (SS) agar plates or Triple Sugar Iron Agar Medium and add them to the serum, at a ratio of 1:1 to measure agglutination. All manipulations were performed under sterile conditions.