Collection of Samples
One hundred and eleven (111) samples, including fish gill, fish intestine and beef (15 each), chicken and pork (24 each) and mutton (18) were collected from various markets in Aizawl, Mizoram, a hilly state in the North-Eastern region India. The samples were collected aseptically by maintaining cold chain and were processed for microbiological analysis within 24 h. This study was conducted for a period of one year from July 2018 to June 2019.

Isolation of Listeria
For isolation of Listeria, ISO 11290 method was used. Each sample was inoculated aseptically in Half Fraser Broth followed by incubation at 30°C. It was subcultured in Fraser broth, further streaked onto PALCAM agar and incubated at 37°C. The grey-green colonies on PALCAM agar with diffuse black-zone was plated on Tryptone Soya Yeast Extract agar. Gram staining and Biochemical tests comprising catalase test, fermentation and acid production from various sugars, followed by MR/VP tests were performed for identification of the isolates. All the Listeria isolates and the control strain were preserved in nutrient agar stab (with 1.5% agar agar) and 16% glycerol for subsequent analysis. L. monocytogenes ATCC 13832 was utilized as the standard strain.

Confirmation of Listeria by PCR
Listeria were cultured in Brain Heart Infusion Broth for overnight for extraction of genomic DNA using Bacterial DNA isolation kit (Genaid, Taiwan). To confirm the suspected isolates, PCR was performed by amplifying Listeria genus-specific prs gene using specific primer¹⁰ (1^st BASE, Malaysia) (Table 1). To confirm L. monocytogenes isolates, primers specific for the virulence associated hlyA gene¹¹ were self-designed using primer BLAST online tool. For detection of L. innocua, PCR amplification of in9 gene was targeted²⁴. A total volume of 25 ml mixture comprising 12.5 ml of 2X Mastermix, 0.5 ml (10 pmol) each of bi-directional primers, DNA (1 ml) and nuclease-free water (10.5 ml) was prepared for PCR. The amplification of prs, hlyA and in9 genes was done by 5 min. denaturation at 95°C, accompanied by thirty cycles of 95°C (30 sec.), 53°C (30 sec.), 72°C (30 sec.) and final elongation at 72°C (5 min.).

Table (1):
Primers used for identification of Listeria species.

Identification of serogroup
A simplex PCR was used to determine the serogroup using group-specific primers (Table 2). Amplification of ORF2819 was used to identify 1/2b and 3b serovars and amplification of both ORF2819 and OFR2110 to identify 4b, 4d and 4e serovars. This PCR does not differentiate within serovars.

Table (2):
Primers used for identification of L. monocytogenes serogroups.

A 25 µl volume of reaction mixture was prepared for PCR same as prepared for Listeria identification. Listeria monocytogenes (ATCC 13832) serotype 4b was used as the positive control. PCR conditions for amplification were as previously described by Michel et al.¹⁰

Virulence associated gene detection
After confirming the isolates by hlyA gene, a simplex PCR was done to determine the three virulence genes, plcA, plcB and iap (Table 3). The composition of reaction mixture was the same as for the other genes used in this study. PCR conditions for amplification of all the three virulence genes were standardized at denaturation at 95°C (5 min.), followed by thirty cycles of 95°C (30 sec.), 52°C (30 sec.), 72°C (30 sec.) and a final elongation at 72°C (5 min.).

Table (3):
Primers used for detection of virulence-associated genes in L. monocytogenes isolates.