Written by – Dr. Lalchhandami Tochhawng, Scientific Officer, MISTIC

(Published in VANGLAINI daily newspaper, 16 June 2020)

Covid-19 vei leh vei lo endik nana khawl hman lar em em RT-PCR hi a lam pum chu ‘Real Time Polymerase Chain Reaction’ a ni a. Tunlai khawvela technology pawimawh berte zinga mi a ni hial awm e. PCR (Polymerase Chain Reaction) khawl hi DNA tihpunna (amplify) atana hman thin a ni. DNA chu ‘Deoxyribonucleic acid’ a ni a, thil nungte inthlah chhawnna atana information pawimawh ber kengtu a ni. PCR hmanga DNA tihpun a nih theih nan hian bawlhlo (chemicals) chi hrang hrang bakah temperature hrang hrang, hunbi neia inthlak hman a ni thin. Real Time-PCR (RT-PCR) bikah hian khawl chhunga thil thleng (reaction) awm chu chawp leh chilhin khawl screen atangin emaw computer hmangin emaw a en nghal zel theih a (hei hi PCR pangai aia a changkanna pawh a ni), hei vang hian a hmingah pawh ‘Real Time’ tih telh a ni.

RT-PCR khawl hmanga COVID-19 en dan:

1) Sample lak

RT-PCR khawl hmanga sample endik tur hi uluk taka lak a ngai a. COVID-19 hi thawkna kawng (respiratory tract) natna a ni a. A hrik SARS-CoV-2 hi hnar, ka leh mit atang te a mihring taksaa lut thei a ni. Chuvang chuan sample endik tur chu hnar chhung emaw hrawk emaw atanga lak a ni thin. Hnar chhung leh hrawkah swab (plastic hmawlh tawite, a hmawra tuihnang hip bet theiin a tuam) in an han hawlh a, hei hian hnar chhung leh hrawk a tui huh/hnang chu a han hip a, sample burah dah luh nghal a ni. Sample burah hian virus chhe tur venna tui a awm a (viral transport medium), vur bawmah temperature hniam, 2-8°C velah vawn that nghal a ni a, a rang thei ang berin endikna hmun (laboratory) panpui tur a ni. Sample hi hun rei zawk chhung vawn that a ngai a nih chuan temperature hniam takah dah a ngai thung.

2) RNA lak chhuah

Sample atanga RNA (Ribonuclei acid) awm zawng zawng chu lak chhuah hmasak a ni phawt a. DNA ni lova RNA lak chhuah a nih zawk nachhan chu SARS-CoV-2 te hian ‘genetic material’ (inthlahchhawn nana an hman) atan RNA zawk an neih vang a ni (DNA an nei ve lo tihna nih chu!). SARS-CoV-2 RNA hi tuamtu kawr thau leh thildang chhungah a awm a. RNA lak chhuah nan hian hemi kawr hi tihchhiat phawt a tul. RNA lak chhuah nan hian bawlhlo (chemicals) leh hmanraw hrang hrang engemawzat a ngai a. A khawih chhung a rei duhin fimkhur a ngai hle bawk. Kan sawi hmaih hauh loh tur chu RNA lak chhuahah hian  virus in a va behchhanna mihring timura RNA pawh a tel. Mihring RNA hi test-na step-a felhlel awm thei venpuitu (control) atan hman tangkai a ni thin.

3) RT-PCR:

RT-PCR khawl chhungah hian thil thleng (reactions) lian tak hlawm hnih a awm a:

1. i) RNA hmanga DNA siam:

SARS-CoV-2-ina nucleic acid a neih hi RNA zawk a nih avangin he an RNA  hmang hian DNA siam chhuah phawt a ngai a. SARS-CoV-2 chauha ‘RNA sequence’ awm, SARS dang leh virus dangtea awm ve lo (specific segment) chauh te chu  entawnin DNA hi siam a ni. [He DNA insiam hi RNA entawn chunga siam a nih avangin cDNA (complimentary DNA) tih kher a ni thin].  cDNA siam nan hian enzyme pawimawh em em ‘reverse transcriptase’ hman a ni a, chuvang chuan he step pawh hi Reverse Transcription PCR tiin an sawi bawk thin. Tin, SARS-CoV-2 RNA chhunga kan duh lai te thlan chhuah nan hian ‘primer’ hman a ni a. Primer chu RNA kan entawn tur lai te chauh mil tura siam nucleic acid sequence tawite a ni.

Virus RNA chu DNA a siam a ni ang tho hian sample-a mihring RNA awm chu primer bik hmanga DNA a siam a ni ve a, control atana hman thin a ni.

Sample positive tur chuan virus RNA atangin DNA hi a insiam ngei ngei a ngai a. SARS-CoV-2 hrik kai lo a nih chuan sample-ah chuan virus RNA a tel lova, chuvang chuan virus RNA atanga DNA insiam tur a awm lovang.

1. ii) DNA tihpun:

RT-PCR hian SARS-CoV-2 DNA (RNA hmanga siam) chu a tipung a. DNA a tihpun rual hian RT-PCR khawl screen emaw computer emaw atanga hmuh theih nghal turin signal a lo lang thin. DNA insiam pung vak a awm chuan sample chu SARS-CoV-2 kai (positive) a ni tih a lantir. Hemi signal a rawn lan theihna tur hian bawlhlo pawimawh tak ‘flourescent probe’ (thim zinga eng thei rawng) hman a ni a. Hemi probe hian DNA a insiam pun apiangin signal a pe chhuak ta thin a ni.  DNA insiam a tam poh leh signal a sang thin.

Test kalpui dan

RT-PCR khawla sample dah luh nan hian 96-well plate hman a ni a. He plate hi kil li nei, kutphah tiat vela hlai (rectangle), khuar (well) te reuh te te 96 nei a ni. A khuar ah hian tui 0.2 ml (200µl) vel lek a leng a. Khuar hi 96 awm mah se, khuar zawng zawng hi sample-in hnawhkhah a ni lo. RT-PCR in hna a thawh chhunga felhlel leh dik lo awm thei hrang hrang venpui turin khuar thenkhat hi sample ni lo thildang hmanga hnawhkhah a ni.

Step1: Screening

Screening atan hian pooled testing hman a ni. Khuar pakhat zelah hian sample panga, mihring panga ami chhunkhawm a ni a, chung sample chhunkhawm te result chu a hlawmin tum khatah RT-PCR khawlin a rawn pe chhuak thin. Result hi a negative a nih chuan sample pangate chu an negative vek tihna a ni a. A positive erawh chuan sample panga zingah hian positive an awm tihna a ni thung.

Step 2: Validation (ennawn lehna)

Screening-a positive awmna khuar hi a bika ennawn leh thin a ni a. Sample panga inpawlh atang khan tu ber nge positive tih finfiah a ni. Hemi atan hian sample hi inpawlh (pool) tawh lovin khuar pakhat zelah a mal mal in RT-PCR khawlah ennawn leh a ni thin.

Test-naa harsatna awm theite:

1. i) RNA lak chhuah hi sample tam thamah chuan hun leh tha sen a hautak duh hle. (Automated RNA extractor thar hian thawktute hun leh tha senral nasa taka a chhawk beisei a ni)
2. ii) Screening-ah positive an tam chuan validation tur a tam zel a, a mal mala tihnawn leh vek angaih thin avangin sample khawih chhung a rei phah.

iii) Sample lak hi uluk viau a ngai a, sample quality a that loh chuan RNA lak chhuah ah harsatna a thlen thei.

Note: A tlangpuiin plate pakhata khuar 92 hi sample chhunkhawm (pooled sample) dah nan hman thin a ni. Sample en tur hi viral target (SARS-CoV-2 RNA hmun hnih a mi, ‘primer’) pahnih zela kalpui a nih chuan pooled sample 46 a dah theih tihna a ni a, a bak khuar 4 te hi chu venpuitu atan hman a ni. Pooled sample pakhatah sample 5 chhunkhawm ni ta se, sample 46 dah luh a nih chuan vawi khat test-ah mimal sample 230 a test theih dawn tihna a ni. Sample chhunkhawmte zingah hian negative an tam chuan pooled testing hi a hlawk hle a, positive an tam erawh chuan a mal te te a ennawn leh vek a ngaih avangin test chhung a rei a, khuar pakhata sample pakhat test nen hun hman rei chhung a inthlau em em chuang lo thei.